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Pig-to-nonhuman primate islet xenotransplantation
Transplant Immunology 21 (2009) 81–86
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Transplant Immunology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / t r i m
Review
Pig-to-nonhuman primate islet xenotransplantation Bernhard J. Hering ⁎, Niketa Walawalkar Schulze Diabetes Institute, University of Minnesota, MMC 280, 420 Delaware Street SE, Minneapolis, MN 55455, USA
a r t i c l e
i n f o
Article history: Received 20 February 2009 Received in revised form 1 May 2009 Accepted 5 May 2009 Keywords: Diabetes Islets of Langerhans Xenotransplantation Nonhuman primates
a b s t r a c t Type 1 diabetes continues to present a therapeutic challenge. The restoration of normoglycemia and insulin independence in immunosuppressed type 1 diabetic recipients of human islet allografts has highlighted the potential of cell-based diabetes therapy. The unlimited and on-demand availability of pig islets from healthy, young, living, designated pathogen-free, and potentially genetically modified donors presents unique opportunities for improving the availability and outcomes of islet replacement therapies in diabetes. One of the fundamental prerequisites for initiating clinical research is a favorable benefit-over-harm determination in the stringent preclinical transplant model in nonhuman primates. To date, xenotransplants of pig islet cell therapy products have been reported by 15 institutions in 181 NHPs, including xenotransplants in 72 nondiabetic and 109 diabetic recipients. These studies have demonstrated the feasibility of successful preclinical islet xenotransplantation and have provided insights into the critical events operative in the immune recognition and destruction of islet xenografts in nonhuman primates. Particularly promising is the recent achievement of prolonged insulin independence in this model by means of several distinct islet xenotransplantation products, implantation sites, and immunotherapeutic strategies. Further progress appears likely and the development of suitable source pigs will position the scientific community to translate these findings safely to the clinic. © 2009 Published by Elsevier B.V.
Contents 1. Rationale for islet xenotransplantation . . . . . . . . . . . . . 2. Rationale for preclinical studies in the pig-to-nonhuman primate 3. Design requirements for informative preclinical studies in NHPs 4. Pig islet cell xenograft survival in NHPs . . . . . . . . . . . . 5. Immunological barriers to islet xenograft survival in NHPs . . . 6. Current and future research directions. . . . . . . . . . . . . 7. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . Contributors . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . model. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Excellent reviews on pig-to-primate islet xenotransplantation have been published in recent years [1–3]. After presenting the rationale for islet xenotransplantation, this review will address the need for preclinical studies in nonhuman primates (NHPs), the design requirements for such studies, the graft survival achieved in this model, the immunological barriers to islet xenograft survival in NHPs, and current and future strategies for overcoming these barriers.
⁎ Corresponding author. Tel.: +1 612 626 5697; fax: +1 612 626 5855. E-mail address: [email protected] (B.J. Hering). 0966-3274/$ – see front matter © 2009 Published by Elsevier B.V. doi:10.1016/j.trim.2009.05.001
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1. Rationale for islet xenotransplantation Human islet transplants are emerging as a viable treatment option for patients with type 1 diabetes (T1D) in whom glycemic management is limited by hypoglycemia unawareness and defective hormonal counterregulation [4–7]. Improved islet processing techniques and novel immunotherapeutic strategies lacking diabetogenic and nonimmunosuppressive side effects are expected to lead to further increases in the benefit–risk ratio of human islet allotransplants and, consequently, to increased demand for islet replacement therapy. However, the supply of human islets cannot meet the current demand. Patients who could benefit from islet transplantation include
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i) the 12.5% of patients with a history of T1D of N20 years and 18% with a history of N30 years who are unaware of autonomic warning symptoms of hypoglycemia and at a very high risk of severe and frequent episodes of hypoglycemia, [8] and ii) the 30 to 50% of patients with T1D who are prone to develop devastating microvascular diabetes complications [9]. Given this prevalence of acute and chronic complications, the overall prevalence of T1D (1.5 million in the US) and the incidence of new cases of T1D (30,000 per year in the US), it is apparent that the number of patients in whom islet transplantation should be considered as a therapeutic option largely exceeds the number of suitable deceased human pancreas donors (3000 per year in the US). Pigs could provide an unlimited and ready supply of islet tissue for transplant with no waiting time long before other surrogate islet β-cell sources and in-situ islet regeneration can be developed to the point of clinical applicability. Pig islets could also provide access to beta cell replacement in countries where human deceased donor pancreas and islets transplantations are not practiced for ethical or cultural reasons [10]. It is important to emphasize that pig islets hold 5 distinct additional advantages over human islets. First, the quality of pig islets prepared from suitable donor pigs is consistent and in contrast to cadaver human islets from deceased donors not compromised by comorbidity, senescence, brain death, and cold ischemia injury. Second, the actual (not theoretical) risks of infectious disease transmission associated with xenografting of islets are lower than in human islet allotransplantation if islets are prepared from designated pathogen-free donor pigs. Third, experimental studies suggest that porcine islet xenografts may be resistant to destruction by recurrent autoimmunity [11–14]. Fourth, the on-demand availability of islets from known source pigs permits recipient pretreatment with donor antigen with the goal to induce donor-specific immunologic hyporesponsiveness [15,16]. Finally, genetic modification of source pigs permits stable overexpression of genes encoding cytoprotective and immunomodulatory peptides in donor islets, thereby presenting opportunities for minimizing recipient immunosuppression not available to recipients of human islet allografts. 2. Rationale for preclinical studies in the pig-to-nonhuman primate model Rooted in the moral duties of beneficence (the duty to benefit others) and non-maleficence (the duty not to harm others), a favorable benefit-over-harm determination has long been recognized as one of the fundamental prerequisites for initiating clinical research [17,18]. Thus, clinical research is only permissible if the probable benefits of the research for both individual subjects and society outweigh the possible risks. Subjects participating in clinical research on pig islet cell therapy products are at risk of experiencing adverse events related to the xenograft product, transplant procedure, and immunosuppressive therapy. Because xenografts may additionally expose patients, and possibly society at large, to unknown potential infectious risks [19], one of the guiding principles of the Ethics Committee of the International Xenotransplantation Association is that there should be a relatively high expectation of benefit, based on sound preclinical data, before such risks can be considered acceptable in clinical trials [10,20,21]. For the benefit-over-harm determination to be favorable in view of known and unknown risks, the anticipated benefits must be substantial such as the restoration and maintenance of normoglycemia and insulin independence after islet xenotransplantation. A high expectation of these benefits calls for their previous documentation in a preclinical study that mimics the intended clinical scenario. Old World monkeys (rhesus and cynomolgus macaques) and baboons are the only suitable recipient animals in which i) many immunotherapeutics designed for human trials cross-react in NHPs and ii) the efficacy of immunosuppression and encapsulation strategies can be
assessed with rigor in the presence of a complex and redundant immune system replete with outbred diversity, heterologous immunity, and MHC expression patterns known to present barriers to longterm islet xenograft graft survival in humans [22]. 3. Design requirements for informative preclinical studies in NHPs For a preclinical study in the pig-to-NHP islet xenotransplant model to provide substantial evidence of safety and efficacy in preparation for a subsequent clinical Phase I/II study, the design of the preclinical study must mimic the intended clinical investigation to the greatest extent possible and must be performed under a high regulatory standard [23]. To meet these criteria, the preclinical studies should be performed in diabetic Old World monkeys or baboons. Successful diabetes induction via streptozotocin or total pancreatectomy should be confirmed by 2 or more blood glucose levels being N350 mg/dl, absence of a C-peptide response to intravenous glucose or arginine, and lack of beta cell regeneration in the native pancreas on autopsy. Manufacturing, release criteria, and dose and route of administration of the investigational xenoislet product tested in the preclinical study and in the intended clinical trial should be similar if not identical. All other protocol-regulated treatment products, in particular immunomodulatory agents, should be administered at dose levels and schedules that mimic the intended clinical scenario. Safety assessments should include regular clinical observations, physical examinations, body weights, food consumption, hematology, chemistry, and urine analysis. Posttransplant metabolic assessments should include frequent measurements of fasting and non-fasting blood or capillary glucose levels, monthly HbA1c or glycosylated hemoglobin levels, as well as periodic metabolic tests to determine glucose and porcine C-peptide responses to mixed meals and/or intravenous glucose, and arginine. A sufficient duration of follow-up, within the limitations of the model, is necessary to evaluate adverse side effects related to the investigational xenograft product and accompanying immunomodulatory agents, the reversibility of any abnormal findings as well as the durability of all activity/ efficacy endpoints. The evaluation of the safety and efficacy of retransplants should be incorporated into the study design if the clinical development program includes the potential for more than one islet xenotransplant. A complete necropsy should be conducted on each animal including detailed microscopic examination of the islet xenograft, the host organ, and the pancreas (if present). Studies examining the xenogeneic pathogen-related infection (particularly PERV), immune responses to the xenograft and porcine insulin, and potential associated toxicities should also be included in the study design. Control cohorts should be incorporated into the design of the preclinical study to delineate toxicities due to immunomodulatory drugs and immunoisolation capsules from toxicities due to the xenograft product. These cohorts should not receive pig islet cell products and include i) unmodified animals as well as ii) non-diabetic control animals given immunomodulatory agent(s) and/or immunoisolation capsules. 4. Pig islet cell xenograft survival in NHPs To date, xenotransplants of pig islet cell therapy products have been reported by 15 institutions in 181 NHPs, including xenotransplants in 72 non-diabetic [24–35] (Table 1) and 109 diabetic recipients [25,28,36–50] (Table 2). In addition, one non-diabetic [29] and eight diabetic recipients have received empty microcapsules [46] (Tables 1 and 2). Furthermore, NHPs have received discordant xenogeneic islet cell products prepared from other species such as mouse [51,52], rat [53], and rabbit pancreas [54]; these studies will not be discussed herein.
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Table 1 Pig islet cell xenografts in non-diabetic nonhuman primates. Donor pig age
Recipient
Site of transplantation
Immunosuppression, encapsulation, and/or genetic engineering
Maximum graft survivala
Reference
Adult
Cyno (n = 8)
Intraportal
Baboon (n = 2)
Intraportal
Posttransplant follow-up for 60 min only N 14 d and b 28 d
Bennet et al. [24]
Adult
Adult
Baboon (n = 4), cyno (n = 1) Group 1 (n = 4): cynoGroup 2 (n = 4): Rhesus
Intraportal
None (n = 7) Pretreatment with sCR1 (n = 1) Whole body and thymic irradiation + ATG + EIA + MMF + CsA + CVF + steroids + α-CD154 ATG + CsA or LF-195 + MMF + steroids
2d
Cantarovich et al. [26]
Group 1: CP + CsA + steroids Group 2: ATG + α-IL-2R + CsA; steroids
Group 1: 11 d
Rijkelijkhuizen et al. [27]
Group 2: ATG + α-IL-2R + CsA; steroids
Group 2: 53 d
None Group 1 (n = 12): encapsulation Group 2 (n = 2): no encapsulation Group 3 (n = 1): empty capsules Group 1: none Group 2: CsA + Aza + steroids Group 3: α-CD4 Group 1b: nonimmunosuppressed Group 2b: CsA + steroids + CP or BQR
N 3d Group 1: b180 d
Kirchhof et al. [28] Dufrane et al. [29]
Group 1: b7 d Group 2: N7 d to b35 d Group 3: b14 d Group 1: b7 d Group 2: N40 d (no benefit from CD55 transgenic donors) Group 1: 6 d Group 2: 12 d Group 1: N50 d Group 2: N50 d Group 2: N56 d (very low levels of porcine C-peptide and only non-β cells on histology)
Mandel et al. [30]
Adult
Group 1 (n = 4): renal subcapsularGroup 2: Renal subcapsular (n = 1), portal vein (n = 3) Group 2: renal subcapsular (n = 1), portal vein (n = 3) Intraportal Renal subcapsular
Adult Adult
Group 2 (n = 4): rhesus Rhesus (n = 2) Cyno (n = 15)
Fetal
Cyno (n = 5)
Fetal
Cyno (n = 5)
Group 1 (n = 1): multiple Group 2 (n = 2): omentum Group 3 (n = 2): renal subcapsular Renal subcapsular
Fetal
Cyno (n = 14)
Renal subcapsular
Neonatal
NHP (n 2)
Intraperitoneal
Neonatal
Macaques (n = 7)
Group 1: omentum, renal subcapsular, pancreatic bed, and intraportal Groups 2–3: omentum
Group 1 (n = 8): nonimmunosuppressed Group 2 (n = 6): CsA + DSG Group 1 (n ≥ 1): microencapsulation Group 2 (n ≥ 1): macroencapsulation Group 1 (n = 2): colocalization of islets with Sertoli cells
Buhler et al. [25]
Mandel et al. [31] Mandel [32] Soderlund et al. [33] Elliott et al. [34] Isaac et al. [35]
Group 2 (n = 2): colocalization of islets with Sertoli cells Group 3 (n = 3): Islets alone
Effects of donor pig age, recipient species, and immunotherapeutic strategy on maximum islet xenograft survival. sCR1, soluble complement receptor 1; ATG, anti-thymocyte globulin; EIA, extracorporeal immunoadsorption; MMF, mycophenolate mofetil; CsA, cyclosporine A; LF-195, deoxyspergualin analog; CP, cyclophosphamide; α-IL2R, anti-interleukin 2 receptor antibody; BQR, brequinar; DSG, deoxyspergualin; Aza, azathioprine; CVF, cobra venom factor. a Graft survival determined by histology and/or porcine C-peptide only. b Including CD55 transgenic and nontransgenic controls.
Prolonged restoration of insulin independence after pig islet cell xenotransplantation for N3 months in NHPs with spontaneous, streptozotocin- (chemically-) and pancreatectomy-(surgically-) induced diabetes has been reported independently by five groups [36,39,43–45,47–49], with functional islet xenograft survival exceeding 6 months in several recipients. Porcine C-peptide was positive in the plasma of these recipients and their fasting and non-fasting blood glucose levels were in the normoglycemic to near-normoglycemic range. Several additional studies have shown prolonged partial xenograft function [42,46] and xenograft survival by histological criteria [27,29,31]. Taken together, these promising findings indicate the feasibility of preclinical evaluation of pig islet cell xenotransplant products in the rigorous NHP model and provide a strong rationale for continued development of islet xenotransplantation into a vital treatment option for type 1 diabetes. Perhaps most intriguing is that success has been possible with several distinct xenotransplantation strategies. Long-term insulin independence has been achieved after i) intraportal infusion of wild-type adult and neonatal pig islets in immunosuppressed rhesus and cynomolgus monkeys with chemical and surgical diabetes [39,47,48], ii) intraportal infusion of islets from adult galactose α-1,3 galactose (Gal)-deficient animals transgenic for human membrane cofactor protein (CD46) in streptozotocin-diabetic, immunosuppressed cynomolgus monkeys [43], iii) implantation of embryonic pig pancreatic precursor tissue in streptozotocin-diabetic immunosuppressed cynomolgus monkeys [49], iv) intraperitoneal transplantation of microencapsulated adult islets in nonimmunosuppressed, spontaneously diabetic monkeys [36], and v) subcutaneous implanta-
tion of a device comprising a mono-layer of encapsulated adult islets on a collagen matrix in nonimmunosuppressed cynomolgus monkeys [44,45,55]. 5. Immunological barriers to islet xenograft survival in NHPs The preclinical studies listed in Tables 1 and 2 have provided important insights into the immune recognition and effector pathways operative in the pig-to-NHP islet xenotransplant model. The specifics of the xenograft product, microenvironment at the implantation site, and the immunosuppressive regimen significantly influence the mechanisms predominating in rejection of xenogeneic islets. The immunological barriers to survival of intraportally transplanted wildtype pig islets in NHPs are briefly reviewed below. The demonstration by several groups of pig islet xenograft survival for weeks and months in monkeys indicates that these cellular xenografts do not undergo hyperacute rejection as observed in vascularized organ transplants (Tables 1 and 2). Furthermore, in monkeys undergoing pig islet xenograft rejection, neither increases in Galspecific IgG or IgM antibody levels nor Gal-specific (isolectin B4) or IgG or IgM staining with associated C9 deposition on islets were observed [39]. Also, adult wild-type pig islets were not more susceptible to early posttransplant graft loss than islets from GT-KO [41] or CD55 transgenic pigs [31]. Two unique features of cultured islet cell xenografts explain their escape from hyperacute rejection without requiring donor Gal epitope elimination or recipient anti-Gal antibody manipulation. First, the Gal epitope is expressed on only 5% of adult and 11% of in vitro-matured neonatal pig islet cells [56]. Second, very
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Table 2 Pig islet cell xenografts in diabeticaa nonhuman primates. Donor pig age
Recipients (n)
Adult
Site of transplantation
Immunosuppression, encapsulation, and/or genetic engineering
Maximum graft survival
Reference
Group 1 (n = 7): encapsulation Group 2 (n = 2): no encapsulation
Group 1: 803 d Group 2: 9 d
Sun et al. [36]
Adult Adult
Intraperitoneal Spontaneously diabetic macaques (n = 9) Baboon (n = 3) Intraportal Rhesus (n = 6) Intraportal
ATG + CsA + Aza None
b2 d N3 d
Adult
Cyno (n = 7)
Renal subcapsular
Adult
Cyno (n = 12)
Intraportal
Group 1 (n = 3): wild type Group 2 (n = 4): GnT-III transgenic pigs Group 1 (n = 3): α-IL-2R + FTY720 + everolimus-CD154 + FTY720 + everolimus Group 3 (-CD154 + FTY720 + everolimus + Leflunomide Group 2 (n = 4): α-IL-2R + α-CD154 + FTY720 + everolimus Group 3 (n = 5): α-IL-2R + α-CD154 + FTY720 + everolimus + leflunomide α-CD25 + α-CD154 + SRL + belatacept Group 1 (n = 2): ATG + Tac ± SRL ± α-CD20 Group 2 (n = 4)b: ATG + α-CD154 + MMF (or ATG + Tac + SRL) + CVF Group 3 (n = 4)c: ATG + α-CD154 + MMF + DS ATG + α-CD154 + MMF + DSd ATG + α-CD154 + MMF; GalT-KO, CD46 transgenic islets Encapsulation (islet multi- and mono-layers)
Group 1: 3 d Group 2: 5 d Group 1: 45 d
Buhler et al. [25] Kirchhof et al. [28] Hardstedt et al. [37] Komoda et al. [38]
Adult Adult
Rhesus (n = 5) Cyno (n = 10)
Intraportal Intraportal
Adult Adult
Cyno (n = 9) Cyno (n = 3)
Intraportal Intraportal
Adult
Cyno (n = 8)
Neonatal
Cyno (n = 16)e
Neonatal
Rhesus (n = 9)
Group 1 (n = 4): renal subcapsular Group 2 (n = 4): subcutaneous with islet mono-layer device Intraperitoneal Group 1 (n = 8): encapsulation + nicotinamide Group 2 (n = 8): empty capsules Intraportal Group 1 (n = 2): no immunosuppression Group 2 (n = 7): α-IL2R + α-CD154 + SRL + belatacept Intraportal α-IL-2R + α-CD40 + belatacept + SRL Omentum ATG + α-CD20 + α-IL-2R + CTL A4-Ig + FTY720 + everolimus Mesentery None
Neonatal Rhesus (n = 6) Embryonic Cyno (n = 3) (E42) Embryonic Rhesus (n = 3) (E28)
Hering et al. [39]
Group 2: N 187 d Group 3: N 158 d N76 d Group 1: b 5 d Group 2: Partial function for N58 d Group 3: 5 d Partial function for N60 d N100 d
Cardona et al. [40] Rood et al. [41]
180 d (subcutaneous islet mono-layer device)
Gianello and Dufrane [44,45]
N252 d (n = 1), partial function for N168 df Group 1: 5 d Group 2: N 260 d
Elliott et al. [46]
N187 d N393 d
Russell et al. [48] Hecht et al. [49]
Casu et al. [42] Ayares et al. [43]
Cardona et al. [47]
Possibly prolonged survival Rogers et al. [50] of individual cells with beta cell morphology
Effects of donor pig age, recipient species, and immunotherapeutic strategy on maximum islet xenograft survival. ATG, anti-thymocyte globulin; CsA, cyclosporine A; Aza, azathioprine; GnT-III, n-acetylglucosaminyltransferase-III; α-IL2R, anti-interleukin 2 receptor antibody; SRL, sirolimus; Tac, tacrolimus; MMF, mycophenolate mofetil; DS, dextran sulphate; CVF, cobra venom factor; GalT-KO, galactose α-1,3 galactose-transferase knockout; CD46, membrane cofactor protein. a Diabetes was induced by intravenous streptozotocin or total pancreatectomy unless otherwise stated. b Including one islet graft from a GT-KO donor pig; up to 80,000 IE/kg. c Including two islet grafts from GT-KO donor pigs; up to 80,000 IE/kg. d Including 5 islet grafts from GT-KO donor pigs. e All subjects received a retransplant 3 months after the first. f Based on reduced insulin requirements compared with control group; no direct evidence of graft function (i.e. porcine C-peptide or histology shown).
few donor islet endothelial cells survive pretransplant culture and transplanted islets are mainly revascularized by recipient endothelial cells [57]. Therefore, in contrast to whole organ xenotransplants, Galspecific antibodies do not seem to have a major role in the rejection of pig islet xenografts in NHPs. Early loss of intraportally transplanted pig islet cells is likely caused by the instant blood-mediated inflammatory reaction (IBMIR) [24,58]. This antigen nonspecific reaction is initiated when islet surfaces are directly exposed to blood and involves the activation of platelets, coagulation and complement cascades, and the infiltration of neutrophils [59]. A detailed histopathological study examining the immune activation pathways in the first 3 d after intraportal transplantation of adult pig islets lodged in the liver of nonimmunosppressed rhesus monkeys showed cell destruction associated with deposition of complement components, platelet accumulation, and neutrophil infiltration at 12 and 24 h posttransplant [28], features consistent with IBMIR. The restoration of normoglycemia and insulin independence after intraportal infusion of 25,000 adult [39] and 50,000 neonatal [47] islet equivalents/kg in monkeys in the apparent absence
of specific treatment for IBMIR does not rule out the possibility of a vigorous IBMIR; it rather suggests that effective targeting of IBMIR holds promise for minimizing the critical islet dose and possibly also the requirements for immunosuppression through prevention of IBMIR-mediated augmentation of the adaptive immune response [59]. The adaptive immune response to intraportal pig islet xenografts in NHPs is largely T-cell dependent and T-cell mediated. Intragraft analysis of transcript levels in nonimmunosuppressed rhesus monkeys demonstrated that CXCR3, with ligands IP-10 and Mig, mediate early T-cell recruitment in acute islet xenograft rejection in NHPs [37]. Rejection, when present in monkeys immunosuppressed with basiliximab, FTY720, and everolimus, was associated with increases in serum antipig IgG non-Gal antibodies, highly significant increases in the number of circulating, IFN-g-secreting, donor-reactive T cells activated by the indirect pathway, and both peri- and intraislet infiltration by CD4+ and CD8+ T cells, by macrophages, and, occasionally, by CD20+ B cells. Neutrophils were rarely observed; natural killer cells and eosinophils were absent [39]. Notably, the demonstration of cell-mediated rejection of islet xenografts between days 25 and 45 in cynomolgus monkeys on
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this regimen [39], which prevented islet allograft rejection in the same monkey species [60], indicates that islet xenograft rejection involves cellular mechanisms that are not predominant in islet allograft rejection. Cytokines and antibodies are expected effectors of islet damage when indirect pathway activation predominates; MHCrestricted, cytolytic mechanisms are expected to contribute to islet graft rejection if T cells are able to respond directly to pig MHC antigens. The actual effector mechanisms remain to be defined.
881) and the National Institutes of Health (U19 AI067151). BJH is the holder of the Eunice L. Dwan Chair in Diabetes Research and the McKnight Presidential Chair in Transplantation Science. We thank Dr. Henk-Jan Schuurman for providing very helpful comments. We also like to acknowledge the instrumental contributions to this work made by our collaborators over the years.
6. Current and future research directions
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A comparison of organ cultured fetal pancreas allo-, iso-, and xenografts (pig) in non-immunosuppressed non-obese diabetic mice. Am J Pathol 1995;147(3):834–44. [12] Simeonovic CJ, Wilson JD. Xenotransplantation of fetal pig proislets in anti-CD4treated diabetic NOD/Lt mice. Transplant Proc 1992;24(5):2287–8. [13] Guo Z, Wu T, Kirchhof N, Mital D, Williams JW, Azuma M, et al. Immunotherapy with nondepleting anti-CD4 monoclonal antibodies but not CD28 antagonists protects islet graft in spontaneously diabetic nod mice from autoimmune destruction and allogeneic and xenogeneic graft rejection. Transplantation 2001;71(11):1656–65. [14] Koulmanda M, Qipo A, Smith RN, Auchincloss Jr H. Pig islet xenografts are resistant to autoimmune destruction by non-obese diabetic recipients after anti-CD4 treatment. Xenotransplantation 2003;10(2):178–84. [15] Appel MC, Banuelos SJ, Greiner DL, Shultz LD, Mordes JP, Rossini AA. Prolonged survival of neonatal porcine islet xenografts in mice treated with a donor-specific transfusion and anti-CD154 antibody. Transplantation 2004;77(9):1341–9. [16] Morelli AE. The immune regulatory effect of apoptotic cells and exosomes on dendritic cells: its impact on transplantation. Am J Transplant 2006;6(2):254–61. [17] Emanuel EJ, Wendler D, Grady C. What makes clinical research ethical? JAMA 2000;283(20):2701–11. [18] H.Y. Vanderpool. Informed consent and xenotransplantation clinical trials. Xenotransplantation; in press. [19] Fishman JA, Patience C. Xenotransplantation: infectious risk revisited. Am J Transplant 2004;4(9):1383–90. [20] Sykes M, d'Apice A, Sandrin M. Position paper of the Ethics Committee of the International Xenotransplantation Association. Transplantation 2004;78(8):1101–7. [21] Sykes M, Cozzi E. Xenotransplantation of pig islets into Mexican children: were the fundamental ethical requirements to proceed with such a study really met? Eur J Endocrinol 2006;154(6):921–2. [22] Kean LS, Gangappa S, Pearson TC, Larsen CP. Transplant tolerance in non-human primates: progress, current challenges and unmet needs. Am J Transplant 2006;6 (5 Pt 1):884–93. [23] U.S.Department of Health and Human Services, Food and Drug Administration, (CBER). Guidance for industry: source animal, product, preclinical and clinical issues concerning the use of xenotransplantation products in humans; 2003. Ref Type: Electronic Citation http://www.fda.gov/cber/gdins/clinxeno.htm. [24] Bennet W, Sundberg B, Lundgren T, Tibell A, Groth CG, Richards A, et al. Damage to porcine islets of Langerhans after exposure to human blood in vitro, or after intraportal transplantation to cynomologus monkeys: protective effects of sCR1 and heparin [see comments]. Transplantation 2000;69(5):711–9. [25] Buhler L, Deng S, O'Neil J, Kitamura H, Koulmanda M, Baldi A, et al. Adult porcine islet transplantation in baboons treated with conventional immunosuppression or a non-myeloablative regimen and CD154 blockade. Xenotransplantation 2002;9 (1):3–13. [26] Cantarovich D, Blancho G, Potiron N, Jugeau N, Fiche M, Chagneau C, et al. Rapid failure of pig islet transplantation in non human primates. Xenotransplantation 2002;9(1):25–35.
While the recent unprecedented accomplishments in preclinical pig-to-NHP islet cell xenotransplantation hold great promise and may have far-reaching implications, they also show important limitations. However, these limitations do not appear insurmountable. Investigators seeking to develop clinically applicable immunosuppressive protocols focus their efforts on the evaluation of immunosuppressive agents that could substitute for anti-CD154 antibodies. These antibodies are a critical component of three protocols that were effective in preventing the rejection of adult pig islets in monkeys [39,43,47]; however, the clinical development of these antibodies was stopped due to severe thromboembolic risks [61]. Preliminary results suggest that antagonistic anti-CD40 monoclonal antibodies could be equally effective when combined with IL-2R antibodies, CD28 antagonists, and sirolimus [48]. Several other immunosuppressants, both investigational and approved, are currently being investigated [62]. Ideally, and in view of the on-demand availability of xenogeneic islet tissue, immunosuppressive drugs should be selected based on their ability to facilitate negative vaccination strategies involving the pretransplant administration of donor antigen to the quiescent immune system of the recipient [63,64]. A large number of islet product-directed strategies are currently under investigation to allow minimization of immunosuppressive requirements. These include the procurement of presumably less immunogenic embryonic pancreatic precursor tissue [49,65], pretransplant islet culture in the presence of mitomycin-C to upregulate TGF-β production of islets [66], surface heparinization [67] or pegylation [68,69] of islets, transfection islets with immunorepellant SDF-1 [70], transgenic islets overexpressing complement regulatory proteins [43,71] and immunoisolation [36,45,46], to name a few. Ultimately, all these strategies share the central objective of creating an immunoprivileged microenvironment at the islet implantation site and/or draining lymph node. It seems therefore desirable to pursue these concepts as part of a multifunctional design that also integrates the local and controlled release of immunoregulatory, proangiogenic, and cytoprotective factors, separate or together with microspheres, nanoparticles, and bioscaffolds [72–75]. 7. Conclusions Building on the remarkable recent progress in the stringent preclinical pig-to-NHP model, research teams are currently pursuing several tantalizing concepts in the promising field of islet xenotransplantation. The simultaneous development of suitable source pigs will position the scientific community to translate these findings safely to the clinic to ultimately provide tangible benefits for patients and their families who are unfairly burdened by type 1 diabetes. Contributors BJH wrote the paper; NW contributed to the compilation of data summarized in Tables 1 and 2. Acknowledgments The work presented in this summary was supported in part by grants from the Juvenile Diabetes Research Foundation (JDRF PPG #21-2006-
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Normalization of diabetes in spontaneously diabetic cynomologus monkeys by xenografts of microencapsulated porcine islets without immunosuppression. J Clin Invest 1996;98(6):1417–22. [37] Hardstedt M, Finnegan CP, Kirchhof N, Hyland KA, Wijkstrom M, Murtaugh MP, et al. Post-transplant upregulation of chemokine messenger RNA in non-human primate recipients of intraportal pig islet xenografts. Xenotransplantation 2005;12:293–302. [38] Komoda H, Miyagawa S, Omori T, Takahagi Y, Murakami H, Shigehisa T, et al. Survival of adult islet grafts from transgenic pigs with N-acetylglucosaminyltransferase-III (GnT-III) in cynomolgus monkeys. Xenotransplantation 2005;12 (3):209–16. [39] Hering BJ, Wijkstrom M, Graham ML, Hardstedt M, Aasheim TC, Jie T, et al. Prolonged diabetes reversal after intraportal xenotransplantation of wild-type porcine islets in immunosuppressed nonhuman primates. Nat Med 2006;12(3):301–3. [40] Cardona K, Milas Z, Strobert E, Cano J, Jiang W, Safley SA, et al. Engraftment of adult porcine islet xenografts in diabetic nonhuman primates through targeting of costimulation pathways. Am J Transplant 2007;7(10):2260–8. [41] Rood PP, Bottino R, Balamurugan AN, Smetanka C, Ayares D, Groth CG, et al. Reduction of early graft loss after intraportal porcine islet transplantation in monkeys. Transplantation 2007;83(2):202–10. [42] Casu A, Bottino R, Balamurugan AN, Hara H, van der Windt DJ, Campanile N, et al. Metabolic aspects of pig-to-monkey (Macaca fascicularis) islet transplantation: implications for translation into clinical practice. Diabetologia 2008;51(1):120–9. [43] Ayares D, Phelps C, Vaught T, Ball S. Genetic engineering of pigs for improved transplant outcomes. Xenotransplantation 2007;14(5):428. [44] Gianello P, Dufrane D. Encapsulation of pig islets by alginate matrix to correct streptozotocin-induced diabetes in primates without immunosuppression. Xenotransplantation 2007;14(5):441. [45] Gianello P, Dufrane D. [Correction of a diabetes mellitus type 1 on primate with encapsulated islet of pig pancreatic transplant]. Bull Mem Acad R Med Belg 2007;162(10–12):439–49. [46] Elliott RB, Escobar L, Tan PL, Garkavenko O, Calafiore R, Basta P, et al. Intraperitoneal alginate-encapsulated neonatal porcine islets in a placebo-controlled study with 16 diabetic cynomolgus primates. Transplant Proc 2005;37(8):3505–8. [47] Cardona K, Korbutt GS, Milas Z, Lyon J, Cano J, Jiang W, et al. Long-term survival of neonatal porcine islets in nonhuman primates by targeting costimulation pathways. Nat Med 2006;12(3):304–6. [48] Russell MC, Cardona K, Olivia VL, Korbutt G, Cano J, Jiang W, et al. Engraftment of neonatal porcine islets in diabetic non-human primates by blockade of the CD28/ CD40 costimulatory pathways. Xenotransplantation 2007;14(5):423. [49] Hecht G, Eventov-Friedman S, Rosen C, Shezen E, Tchorsh D, Aronovich A, et al. Embryonic pig pancreatic tissue for the treatment of diabetes in a nonhuman primate model. Proc Natl Acad Sci USA 2009 [May 11, Electronic publication ahead of print], doi:10.1073/pnas.0812253106. [50] Rogers SA, Chen F, Talcott MR, Faulkner C, Thomas JM, Thevis M, et al. Long-term engraftment following transplantation of pig pancreatic primordia into non-immunosuppressed diabetic rhesus macaques. Xenotransplantation 2007;14(6):591–602.
[51] Hamelmann W, Gray DW, Cairns TD, Ozasa T, Ferguson DJ, Cahill A, et al. Immediate destruction of xenogeneic islets in a primate model. Transplantation 1994;58 (10):1109–14. [52] Badet L, Titus TT, McShane P, Chang LW, Song Z, Ferguson DJ, et al. Transplantation of mouse pancreatic islets into primates—in vivo and in vitro evaluation. Transplantation 2001;72(12):1867–74. [53] Tze WJ, Tai J. Xenotransplantation of rat pancreatic endocrine cells in spontaneous and streptozotocin-induced diabetic monkeys. Transplant Proc 1989;21(1 Pt 3):2736–8. [54] Hamelmann W, Ozasa T, Cairns T, Welsh K, Gray DW. Xenotransplantation of islets across a strong species barrier (rabbit to cynomolgus monkey). Transplant Proc 1994;26(3):1097. [55] Dufrane D, Mourad M, van SM, Goebbels RM, Gianello P. Regeneration of abdominal wall musculofascial defects by a human acellular collagen matrix. Biomaterials 2008;29(14):2237–48. [56] Rayat GR, Rajotte RV, Hering BJ, Binette TM, Korbutt GS. In vitro and in vivo expression of Galalpha-(1,3)Gal on porcine islet cells is age dependent. J Endocrinol 2003;177 (1):127–35. [57] Nyqvist D, Koehler M, Wahlstedt H, Berggren PO. Donor islet enothelial cells participate in formation of functional vessels within pancreatic islet grafts. Diabetes 2005;54:2287–93. [58] Bennet W, Sundberg B, Groth CG, Brendel MD, Brandhorst D, Brandhorst H, et al. Incompatibility between human blood and isolated islets of Langerhans: a finding with implications for clinical intraportal islet transplantation? Diabetes 1999;48:1907–14. [59] Nilsson B. The instant blood-mediated inflammatory reaction in xenogeneic islet transplantation. Xenotransplantation 2008;15(2):96–8. [60] Wijkstrom M, Kenyon NS, Kirchhof N, Kenyon NM, Mullon C, Lake P, et al. Islet allograft survival in nonhuman primates immunosuppressed with basiliximab, RAD, and FTY720. Transplantation 2004;77(6):827–35. [61] Andre P, Prasad KS, Denis CV, He M, Papalia JM, Hynes RO, et al. CD40L stabilizes arterial thrombi by a beta3 integrin-dependent mechanism. Nat Med 2002;8 (3):247–52. [62] Gangappa S, Larsen CP. Immunosuppressive protocols for pig-to-human islet transplantation: lessons from pre-clinical non-human primate models. Xenotransplantation 2008;15(2):107–11. [63] Quezada SA, Jarvinen LZ, Lind EF, Noelle RJ. CD40/CD154 interactions at the interface of tolerance and immunity. Annu Rev Immunol 2004;22:307–28. [64] Morelli AE, Thomson AW. Tolerogenic dendritic cells and the quest for transplant tolerance. Nat Rev Immunol 2007;7(8):610–21. [65] Eventov-Friedman S, Tchorsh D, Katchman H, Shezen E, Aronovich A, Hecht G, et al. Embryonic pig pancreatic tissue transplantation for the treatment of diabetes. PLoS Med 2006;3(7):e215. [66] Gunji T, Saito T, Sato Y, Matsuyama S, Ise K, Kimura T, et al. Mitomycin-C treatment followed by culture produces long-term survival of islet xenografts in a rat-to mouse model. Cell Transplant 2008;17(6):619–29. [67] Cabric S, Sanchez J, Lundgren T, Foss A, Felldin M, Kallen R, et al. Islet surface heparinization prevents the instant blood-mediated inflammatory reaction in islet transplantation. Diabetes 2007;56(8):2008–15. [68] Yun LD, Hee NJ, Byun Y. Functional and histological evaluation of transplanted pancreatic islets immunoprotected by PEGylation and cyclosporine for 1 year. Biomaterials 2007;28(11):1957–66. [69] Wilson JT, Cui W, Chaikof EL. Layer-by-layer assembly of a conformal nanothin PEG coating for intraportal islet transplantation. Nano Lett 2008;8(7):1940–8. [70] Papeta N, Chen T, Vianello F, Gererty L, Malik A, Mok YT, et al. Long-term survival of transplanted allogeneic cells engineered to express a T cell chemorepellent. Transplantation 2007;83(2):174–83. [71] d'Apice AJ, Cowan PJ. Gene-modified pigs. Xenotransplantation 2008;15(2):87–90. [72] Blomeier H, Zhang X, Rives C, Brissova M, Hughes E, Baker M, et al. Polymer scaffolds as synthetic microenvironments for extrahepatic islet transplantation. Transplantation 2006;82(4):452–9. [73] Salvay DM, Rives CB, Zhang X, Chen F, Kaufman DB, Lowe Jr WL, et al. Extracellular matrix protein-coated scaffolds promote the reversal of diabetes after extrahepatic islet transplantation. Transplantation 2008;85(10):1456–64. [74] Stendahl JC, Wang LJ, Chow LW, Kaufman DB, Stupp SI. Growth factor delivery from self-assembling nanofibers to facilitate islet transplantation. Transplantation 2008;86(3):478–81. [75] Berman DM, O'Neil JJ, Coffey LC, Chaffanjon PC, Kenyon NM, Ruiz Jr P, et al. Longterm survival of nonhuman primate islets implanted in an omental pouch on a biodegradable scaffold. Am J Transplant 2009;9(1):91–104.
Contents lists available at ScienceDirect
Transplant Immunology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / t r i m
Review
Pig-to-nonhuman primate islet xenotransplantation Bernhard J. Hering ⁎, Niketa Walawalkar Schulze Diabetes Institute, University of Minnesota, MMC 280, 420 Delaware Street SE, Minneapolis, MN 55455, USA
a r t i c l e
i n f o
Article history: Received 20 February 2009 Received in revised form 1 May 2009 Accepted 5 May 2009 Keywords: Diabetes Islets of Langerhans Xenotransplantation Nonhuman primates
a b s t r a c t Type 1 diabetes continues to present a therapeutic challenge. The restoration of normoglycemia and insulin independence in immunosuppressed type 1 diabetic recipients of human islet allografts has highlighted the potential of cell-based diabetes therapy. The unlimited and on-demand availability of pig islets from healthy, young, living, designated pathogen-free, and potentially genetically modified donors presents unique opportunities for improving the availability and outcomes of islet replacement therapies in diabetes. One of the fundamental prerequisites for initiating clinical research is a favorable benefit-over-harm determination in the stringent preclinical transplant model in nonhuman primates. To date, xenotransplants of pig islet cell therapy products have been reported by 15 institutions in 181 NHPs, including xenotransplants in 72 nondiabetic and 109 diabetic recipients. These studies have demonstrated the feasibility of successful preclinical islet xenotransplantation and have provided insights into the critical events operative in the immune recognition and destruction of islet xenografts in nonhuman primates. Particularly promising is the recent achievement of prolonged insulin independence in this model by means of several distinct islet xenotransplantation products, implantation sites, and immunotherapeutic strategies. Further progress appears likely and the development of suitable source pigs will position the scientific community to translate these findings safely to the clinic. © 2009 Published by Elsevier B.V.
Contents 1. Rationale for islet xenotransplantation . . . . . . . . . . . . . 2. Rationale for preclinical studies in the pig-to-nonhuman primate 3. Design requirements for informative preclinical studies in NHPs 4. Pig islet cell xenograft survival in NHPs . . . . . . . . . . . . 5. Immunological barriers to islet xenograft survival in NHPs . . . 6. Current and future research directions. . . . . . . . . . . . . 7. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . Contributors . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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Excellent reviews on pig-to-primate islet xenotransplantation have been published in recent years [1–3]. After presenting the rationale for islet xenotransplantation, this review will address the need for preclinical studies in nonhuman primates (NHPs), the design requirements for such studies, the graft survival achieved in this model, the immunological barriers to islet xenograft survival in NHPs, and current and future strategies for overcoming these barriers.
⁎ Corresponding author. Tel.: +1 612 626 5697; fax: +1 612 626 5855. E-mail address: [email protected] (B.J. Hering). 0966-3274/$ – see front matter © 2009 Published by Elsevier B.V. doi:10.1016/j.trim.2009.05.001
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1. Rationale for islet xenotransplantation Human islet transplants are emerging as a viable treatment option for patients with type 1 diabetes (T1D) in whom glycemic management is limited by hypoglycemia unawareness and defective hormonal counterregulation [4–7]. Improved islet processing techniques and novel immunotherapeutic strategies lacking diabetogenic and nonimmunosuppressive side effects are expected to lead to further increases in the benefit–risk ratio of human islet allotransplants and, consequently, to increased demand for islet replacement therapy. However, the supply of human islets cannot meet the current demand. Patients who could benefit from islet transplantation include
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i) the 12.5% of patients with a history of T1D of N20 years and 18% with a history of N30 years who are unaware of autonomic warning symptoms of hypoglycemia and at a very high risk of severe and frequent episodes of hypoglycemia, [8] and ii) the 30 to 50% of patients with T1D who are prone to develop devastating microvascular diabetes complications [9]. Given this prevalence of acute and chronic complications, the overall prevalence of T1D (1.5 million in the US) and the incidence of new cases of T1D (30,000 per year in the US), it is apparent that the number of patients in whom islet transplantation should be considered as a therapeutic option largely exceeds the number of suitable deceased human pancreas donors (3000 per year in the US). Pigs could provide an unlimited and ready supply of islet tissue for transplant with no waiting time long before other surrogate islet β-cell sources and in-situ islet regeneration can be developed to the point of clinical applicability. Pig islets could also provide access to beta cell replacement in countries where human deceased donor pancreas and islets transplantations are not practiced for ethical or cultural reasons [10]. It is important to emphasize that pig islets hold 5 distinct additional advantages over human islets. First, the quality of pig islets prepared from suitable donor pigs is consistent and in contrast to cadaver human islets from deceased donors not compromised by comorbidity, senescence, brain death, and cold ischemia injury. Second, the actual (not theoretical) risks of infectious disease transmission associated with xenografting of islets are lower than in human islet allotransplantation if islets are prepared from designated pathogen-free donor pigs. Third, experimental studies suggest that porcine islet xenografts may be resistant to destruction by recurrent autoimmunity [11–14]. Fourth, the on-demand availability of islets from known source pigs permits recipient pretreatment with donor antigen with the goal to induce donor-specific immunologic hyporesponsiveness [15,16]. Finally, genetic modification of source pigs permits stable overexpression of genes encoding cytoprotective and immunomodulatory peptides in donor islets, thereby presenting opportunities for minimizing recipient immunosuppression not available to recipients of human islet allografts. 2. Rationale for preclinical studies in the pig-to-nonhuman primate model Rooted in the moral duties of beneficence (the duty to benefit others) and non-maleficence (the duty not to harm others), a favorable benefit-over-harm determination has long been recognized as one of the fundamental prerequisites for initiating clinical research [17,18]. Thus, clinical research is only permissible if the probable benefits of the research for both individual subjects and society outweigh the possible risks. Subjects participating in clinical research on pig islet cell therapy products are at risk of experiencing adverse events related to the xenograft product, transplant procedure, and immunosuppressive therapy. Because xenografts may additionally expose patients, and possibly society at large, to unknown potential infectious risks [19], one of the guiding principles of the Ethics Committee of the International Xenotransplantation Association is that there should be a relatively high expectation of benefit, based on sound preclinical data, before such risks can be considered acceptable in clinical trials [10,20,21]. For the benefit-over-harm determination to be favorable in view of known and unknown risks, the anticipated benefits must be substantial such as the restoration and maintenance of normoglycemia and insulin independence after islet xenotransplantation. A high expectation of these benefits calls for their previous documentation in a preclinical study that mimics the intended clinical scenario. Old World monkeys (rhesus and cynomolgus macaques) and baboons are the only suitable recipient animals in which i) many immunotherapeutics designed for human trials cross-react in NHPs and ii) the efficacy of immunosuppression and encapsulation strategies can be
assessed with rigor in the presence of a complex and redundant immune system replete with outbred diversity, heterologous immunity, and MHC expression patterns known to present barriers to longterm islet xenograft graft survival in humans [22]. 3. Design requirements for informative preclinical studies in NHPs For a preclinical study in the pig-to-NHP islet xenotransplant model to provide substantial evidence of safety and efficacy in preparation for a subsequent clinical Phase I/II study, the design of the preclinical study must mimic the intended clinical investigation to the greatest extent possible and must be performed under a high regulatory standard [23]. To meet these criteria, the preclinical studies should be performed in diabetic Old World monkeys or baboons. Successful diabetes induction via streptozotocin or total pancreatectomy should be confirmed by 2 or more blood glucose levels being N350 mg/dl, absence of a C-peptide response to intravenous glucose or arginine, and lack of beta cell regeneration in the native pancreas on autopsy. Manufacturing, release criteria, and dose and route of administration of the investigational xenoislet product tested in the preclinical study and in the intended clinical trial should be similar if not identical. All other protocol-regulated treatment products, in particular immunomodulatory agents, should be administered at dose levels and schedules that mimic the intended clinical scenario. Safety assessments should include regular clinical observations, physical examinations, body weights, food consumption, hematology, chemistry, and urine analysis. Posttransplant metabolic assessments should include frequent measurements of fasting and non-fasting blood or capillary glucose levels, monthly HbA1c or glycosylated hemoglobin levels, as well as periodic metabolic tests to determine glucose and porcine C-peptide responses to mixed meals and/or intravenous glucose, and arginine. A sufficient duration of follow-up, within the limitations of the model, is necessary to evaluate adverse side effects related to the investigational xenograft product and accompanying immunomodulatory agents, the reversibility of any abnormal findings as well as the durability of all activity/ efficacy endpoints. The evaluation of the safety and efficacy of retransplants should be incorporated into the study design if the clinical development program includes the potential for more than one islet xenotransplant. A complete necropsy should be conducted on each animal including detailed microscopic examination of the islet xenograft, the host organ, and the pancreas (if present). Studies examining the xenogeneic pathogen-related infection (particularly PERV), immune responses to the xenograft and porcine insulin, and potential associated toxicities should also be included in the study design. Control cohorts should be incorporated into the design of the preclinical study to delineate toxicities due to immunomodulatory drugs and immunoisolation capsules from toxicities due to the xenograft product. These cohorts should not receive pig islet cell products and include i) unmodified animals as well as ii) non-diabetic control animals given immunomodulatory agent(s) and/or immunoisolation capsules. 4. Pig islet cell xenograft survival in NHPs To date, xenotransplants of pig islet cell therapy products have been reported by 15 institutions in 181 NHPs, including xenotransplants in 72 non-diabetic [24–35] (Table 1) and 109 diabetic recipients [25,28,36–50] (Table 2). In addition, one non-diabetic [29] and eight diabetic recipients have received empty microcapsules [46] (Tables 1 and 2). Furthermore, NHPs have received discordant xenogeneic islet cell products prepared from other species such as mouse [51,52], rat [53], and rabbit pancreas [54]; these studies will not be discussed herein.
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83
Table 1 Pig islet cell xenografts in non-diabetic nonhuman primates. Donor pig age
Recipient
Site of transplantation
Immunosuppression, encapsulation, and/or genetic engineering
Maximum graft survivala
Reference
Adult
Cyno (n = 8)
Intraportal
Baboon (n = 2)
Intraportal
Posttransplant follow-up for 60 min only N 14 d and b 28 d
Bennet et al. [24]
Adult
Adult
Baboon (n = 4), cyno (n = 1) Group 1 (n = 4): cynoGroup 2 (n = 4): Rhesus
Intraportal
None (n = 7) Pretreatment with sCR1 (n = 1) Whole body and thymic irradiation + ATG + EIA + MMF + CsA + CVF + steroids + α-CD154 ATG + CsA or LF-195 + MMF + steroids
2d
Cantarovich et al. [26]
Group 1: CP + CsA + steroids Group 2: ATG + α-IL-2R + CsA; steroids
Group 1: 11 d
Rijkelijkhuizen et al. [27]
Group 2: ATG + α-IL-2R + CsA; steroids
Group 2: 53 d
None Group 1 (n = 12): encapsulation Group 2 (n = 2): no encapsulation Group 3 (n = 1): empty capsules Group 1: none Group 2: CsA + Aza + steroids Group 3: α-CD4 Group 1b: nonimmunosuppressed Group 2b: CsA + steroids + CP or BQR
N 3d Group 1: b180 d
Kirchhof et al. [28] Dufrane et al. [29]
Group 1: b7 d Group 2: N7 d to b35 d Group 3: b14 d Group 1: b7 d Group 2: N40 d (no benefit from CD55 transgenic donors) Group 1: 6 d Group 2: 12 d Group 1: N50 d Group 2: N50 d Group 2: N56 d (very low levels of porcine C-peptide and only non-β cells on histology)
Mandel et al. [30]
Adult
Group 1 (n = 4): renal subcapsularGroup 2: Renal subcapsular (n = 1), portal vein (n = 3) Group 2: renal subcapsular (n = 1), portal vein (n = 3) Intraportal Renal subcapsular
Adult Adult
Group 2 (n = 4): rhesus Rhesus (n = 2) Cyno (n = 15)
Fetal
Cyno (n = 5)
Fetal
Cyno (n = 5)
Group 1 (n = 1): multiple Group 2 (n = 2): omentum Group 3 (n = 2): renal subcapsular Renal subcapsular
Fetal
Cyno (n = 14)
Renal subcapsular
Neonatal
NHP (n 2)
Intraperitoneal
Neonatal
Macaques (n = 7)
Group 1: omentum, renal subcapsular, pancreatic bed, and intraportal Groups 2–3: omentum
Group 1 (n = 8): nonimmunosuppressed Group 2 (n = 6): CsA + DSG Group 1 (n ≥ 1): microencapsulation Group 2 (n ≥ 1): macroencapsulation Group 1 (n = 2): colocalization of islets with Sertoli cells
Buhler et al. [25]
Mandel et al. [31] Mandel [32] Soderlund et al. [33] Elliott et al. [34] Isaac et al. [35]
Group 2 (n = 2): colocalization of islets with Sertoli cells Group 3 (n = 3): Islets alone
Effects of donor pig age, recipient species, and immunotherapeutic strategy on maximum islet xenograft survival. sCR1, soluble complement receptor 1; ATG, anti-thymocyte globulin; EIA, extracorporeal immunoadsorption; MMF, mycophenolate mofetil; CsA, cyclosporine A; LF-195, deoxyspergualin analog; CP, cyclophosphamide; α-IL2R, anti-interleukin 2 receptor antibody; BQR, brequinar; DSG, deoxyspergualin; Aza, azathioprine; CVF, cobra venom factor. a Graft survival determined by histology and/or porcine C-peptide only. b Including CD55 transgenic and nontransgenic controls.
Prolonged restoration of insulin independence after pig islet cell xenotransplantation for N3 months in NHPs with spontaneous, streptozotocin- (chemically-) and pancreatectomy-(surgically-) induced diabetes has been reported independently by five groups [36,39,43–45,47–49], with functional islet xenograft survival exceeding 6 months in several recipients. Porcine C-peptide was positive in the plasma of these recipients and their fasting and non-fasting blood glucose levels were in the normoglycemic to near-normoglycemic range. Several additional studies have shown prolonged partial xenograft function [42,46] and xenograft survival by histological criteria [27,29,31]. Taken together, these promising findings indicate the feasibility of preclinical evaluation of pig islet cell xenotransplant products in the rigorous NHP model and provide a strong rationale for continued development of islet xenotransplantation into a vital treatment option for type 1 diabetes. Perhaps most intriguing is that success has been possible with several distinct xenotransplantation strategies. Long-term insulin independence has been achieved after i) intraportal infusion of wild-type adult and neonatal pig islets in immunosuppressed rhesus and cynomolgus monkeys with chemical and surgical diabetes [39,47,48], ii) intraportal infusion of islets from adult galactose α-1,3 galactose (Gal)-deficient animals transgenic for human membrane cofactor protein (CD46) in streptozotocin-diabetic, immunosuppressed cynomolgus monkeys [43], iii) implantation of embryonic pig pancreatic precursor tissue in streptozotocin-diabetic immunosuppressed cynomolgus monkeys [49], iv) intraperitoneal transplantation of microencapsulated adult islets in nonimmunosuppressed, spontaneously diabetic monkeys [36], and v) subcutaneous implanta-
tion of a device comprising a mono-layer of encapsulated adult islets on a collagen matrix in nonimmunosuppressed cynomolgus monkeys [44,45,55]. 5. Immunological barriers to islet xenograft survival in NHPs The preclinical studies listed in Tables 1 and 2 have provided important insights into the immune recognition and effector pathways operative in the pig-to-NHP islet xenotransplant model. The specifics of the xenograft product, microenvironment at the implantation site, and the immunosuppressive regimen significantly influence the mechanisms predominating in rejection of xenogeneic islets. The immunological barriers to survival of intraportally transplanted wildtype pig islets in NHPs are briefly reviewed below. The demonstration by several groups of pig islet xenograft survival for weeks and months in monkeys indicates that these cellular xenografts do not undergo hyperacute rejection as observed in vascularized organ transplants (Tables 1 and 2). Furthermore, in monkeys undergoing pig islet xenograft rejection, neither increases in Galspecific IgG or IgM antibody levels nor Gal-specific (isolectin B4) or IgG or IgM staining with associated C9 deposition on islets were observed [39]. Also, adult wild-type pig islets were not more susceptible to early posttransplant graft loss than islets from GT-KO [41] or CD55 transgenic pigs [31]. Two unique features of cultured islet cell xenografts explain their escape from hyperacute rejection without requiring donor Gal epitope elimination or recipient anti-Gal antibody manipulation. First, the Gal epitope is expressed on only 5% of adult and 11% of in vitro-matured neonatal pig islet cells [56]. Second, very
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Table 2 Pig islet cell xenografts in diabeticaa nonhuman primates. Donor pig age
Recipients (n)
Adult
Site of transplantation
Immunosuppression, encapsulation, and/or genetic engineering
Maximum graft survival
Reference
Group 1 (n = 7): encapsulation Group 2 (n = 2): no encapsulation
Group 1: 803 d Group 2: 9 d
Sun et al. [36]
Adult Adult
Intraperitoneal Spontaneously diabetic macaques (n = 9) Baboon (n = 3) Intraportal Rhesus (n = 6) Intraportal
ATG + CsA + Aza None
b2 d N3 d
Adult
Cyno (n = 7)
Renal subcapsular
Adult
Cyno (n = 12)
Intraportal
Group 1 (n = 3): wild type Group 2 (n = 4): GnT-III transgenic pigs Group 1 (n = 3): α-IL-2R + FTY720 + everolimus-CD154 + FTY720 + everolimus Group 3 (-CD154 + FTY720 + everolimus + Leflunomide Group 2 (n = 4): α-IL-2R + α-CD154 + FTY720 + everolimus Group 3 (n = 5): α-IL-2R + α-CD154 + FTY720 + everolimus + leflunomide α-CD25 + α-CD154 + SRL + belatacept Group 1 (n = 2): ATG + Tac ± SRL ± α-CD20 Group 2 (n = 4)b: ATG + α-CD154 + MMF (or ATG + Tac + SRL) + CVF Group 3 (n = 4)c: ATG + α-CD154 + MMF + DS ATG + α-CD154 + MMF + DSd ATG + α-CD154 + MMF; GalT-KO, CD46 transgenic islets Encapsulation (islet multi- and mono-layers)
Group 1: 3 d Group 2: 5 d Group 1: 45 d
Buhler et al. [25] Kirchhof et al. [28] Hardstedt et al. [37] Komoda et al. [38]
Adult Adult
Rhesus (n = 5) Cyno (n = 10)
Intraportal Intraportal
Adult Adult
Cyno (n = 9) Cyno (n = 3)
Intraportal Intraportal
Adult
Cyno (n = 8)
Neonatal
Cyno (n = 16)e
Neonatal
Rhesus (n = 9)
Group 1 (n = 4): renal subcapsular Group 2 (n = 4): subcutaneous with islet mono-layer device Intraperitoneal Group 1 (n = 8): encapsulation + nicotinamide Group 2 (n = 8): empty capsules Intraportal Group 1 (n = 2): no immunosuppression Group 2 (n = 7): α-IL2R + α-CD154 + SRL + belatacept Intraportal α-IL-2R + α-CD40 + belatacept + SRL Omentum ATG + α-CD20 + α-IL-2R + CTL A4-Ig + FTY720 + everolimus Mesentery None
Neonatal Rhesus (n = 6) Embryonic Cyno (n = 3) (E42) Embryonic Rhesus (n = 3) (E28)
Hering et al. [39]
Group 2: N 187 d Group 3: N 158 d N76 d Group 1: b 5 d Group 2: Partial function for N58 d Group 3: 5 d Partial function for N60 d N100 d
Cardona et al. [40] Rood et al. [41]
180 d (subcutaneous islet mono-layer device)
Gianello and Dufrane [44,45]
N252 d (n = 1), partial function for N168 df Group 1: 5 d Group 2: N 260 d
Elliott et al. [46]
N187 d N393 d
Russell et al. [48] Hecht et al. [49]
Casu et al. [42] Ayares et al. [43]
Cardona et al. [47]
Possibly prolonged survival Rogers et al. [50] of individual cells with beta cell morphology
Effects of donor pig age, recipient species, and immunotherapeutic strategy on maximum islet xenograft survival. ATG, anti-thymocyte globulin; CsA, cyclosporine A; Aza, azathioprine; GnT-III, n-acetylglucosaminyltransferase-III; α-IL2R, anti-interleukin 2 receptor antibody; SRL, sirolimus; Tac, tacrolimus; MMF, mycophenolate mofetil; DS, dextran sulphate; CVF, cobra venom factor; GalT-KO, galactose α-1,3 galactose-transferase knockout; CD46, membrane cofactor protein. a Diabetes was induced by intravenous streptozotocin or total pancreatectomy unless otherwise stated. b Including one islet graft from a GT-KO donor pig; up to 80,000 IE/kg. c Including two islet grafts from GT-KO donor pigs; up to 80,000 IE/kg. d Including 5 islet grafts from GT-KO donor pigs. e All subjects received a retransplant 3 months after the first. f Based on reduced insulin requirements compared with control group; no direct evidence of graft function (i.e. porcine C-peptide or histology shown).
few donor islet endothelial cells survive pretransplant culture and transplanted islets are mainly revascularized by recipient endothelial cells [57]. Therefore, in contrast to whole organ xenotransplants, Galspecific antibodies do not seem to have a major role in the rejection of pig islet xenografts in NHPs. Early loss of intraportally transplanted pig islet cells is likely caused by the instant blood-mediated inflammatory reaction (IBMIR) [24,58]. This antigen nonspecific reaction is initiated when islet surfaces are directly exposed to blood and involves the activation of platelets, coagulation and complement cascades, and the infiltration of neutrophils [59]. A detailed histopathological study examining the immune activation pathways in the first 3 d after intraportal transplantation of adult pig islets lodged in the liver of nonimmunosppressed rhesus monkeys showed cell destruction associated with deposition of complement components, platelet accumulation, and neutrophil infiltration at 12 and 24 h posttransplant [28], features consistent with IBMIR. The restoration of normoglycemia and insulin independence after intraportal infusion of 25,000 adult [39] and 50,000 neonatal [47] islet equivalents/kg in monkeys in the apparent absence
of specific treatment for IBMIR does not rule out the possibility of a vigorous IBMIR; it rather suggests that effective targeting of IBMIR holds promise for minimizing the critical islet dose and possibly also the requirements for immunosuppression through prevention of IBMIR-mediated augmentation of the adaptive immune response [59]. The adaptive immune response to intraportal pig islet xenografts in NHPs is largely T-cell dependent and T-cell mediated. Intragraft analysis of transcript levels in nonimmunosuppressed rhesus monkeys demonstrated that CXCR3, with ligands IP-10 and Mig, mediate early T-cell recruitment in acute islet xenograft rejection in NHPs [37]. Rejection, when present in monkeys immunosuppressed with basiliximab, FTY720, and everolimus, was associated with increases in serum antipig IgG non-Gal antibodies, highly significant increases in the number of circulating, IFN-g-secreting, donor-reactive T cells activated by the indirect pathway, and both peri- and intraislet infiltration by CD4+ and CD8+ T cells, by macrophages, and, occasionally, by CD20+ B cells. Neutrophils were rarely observed; natural killer cells and eosinophils were absent [39]. Notably, the demonstration of cell-mediated rejection of islet xenografts between days 25 and 45 in cynomolgus monkeys on
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this regimen [39], which prevented islet allograft rejection in the same monkey species [60], indicates that islet xenograft rejection involves cellular mechanisms that are not predominant in islet allograft rejection. Cytokines and antibodies are expected effectors of islet damage when indirect pathway activation predominates; MHCrestricted, cytolytic mechanisms are expected to contribute to islet graft rejection if T cells are able to respond directly to pig MHC antigens. The actual effector mechanisms remain to be defined.
881) and the National Institutes of Health (U19 AI067151). BJH is the holder of the Eunice L. Dwan Chair in Diabetes Research and the McKnight Presidential Chair in Transplantation Science. We thank Dr. Henk-Jan Schuurman for providing very helpful comments. We also like to acknowledge the instrumental contributions to this work made by our collaborators over the years.
6. Current and future research directions
[1] Rood PP, Buhler LH, Bottino R, Trucco M, Cooper DK. Pig-to-nonhuman primate islet xenotransplantation: a review of current problems. Cell Transplant 2006;15 (2):89–104. [2] Cozzi E, Bosio E. Islet xenotransplantation: current status of preclinical studies in the pig-to-nonhuman primate model. Curr Opin Organ Transplant 2008;13(2):155–8. [3] Dufrane D, Gianello P. Pig islet xenotransplantation into non-human primate model. Transplantation 2008;86(6):753–60. [4] Shapiro AMJ, Lakey JRT, Ryan EA, Korbutt GS, Toth E, Warnock GL, et al. Islet transplantation in seven patients with type 1 diabetes mellitus using a glucocorticoid-free immunosuppressive regimen. N Engl J Med 2000;343:230–8. [5] Hering BJ, Kandaswamy R, Ansite JD, Eckman PM, Nakano M, Sawada T, et al. Single-donor, marginal-dose islet transplantation in patients with type 1 diabetes. JAMA 2005;293(7):830–5. [6] Shapiro AM, Ricordi C, Hering BJ, Auchincloss H, Lindblad R, Robertson RP, et al. International trial of the Edmonton protocol for islet transplantation. N Engl J Med 2006;355(13):1318–30. [7] Hogan A, Pileggi A, Ricordi C. Transplantation: current developments and future directions; the future of clinical islet transplantation as a cure for diabetes. Front Biosci 2008;13:1192–205. [8] Pedersen-Bjergaard U, Pramming S, Heller SR, Wallace TM, Rasmussen AK, Jorgensen HV, et al. Severe hypoglycaemia in 1076 adult patients with type 1 diabetes: influence of risk markers and selection. Diabetes Metab Res Rev 2004;20 (6):479–86. [9] Pambianco G, Costacou T, Ellis D, Becker DJ, Klein R, Orchard TJ. The 30-year natural history of type 1 diabetes complications: the Pittsburgh Epidemiology of Diabetes Complications Study experience. Diabetes 2006;55(5):1463–9. [10] Sykes M, d'Apice A, Sandrin M. Position paper of the Ethics Committee of the International Xenotransplantation Association. Xenotransplantation 2003;10 (3):194–203. [11] Mandel TE, Kovarik J, Koulmanda M. A comparison of organ cultured fetal pancreas allo-, iso-, and xenografts (pig) in non-immunosuppressed non-obese diabetic mice. Am J Pathol 1995;147(3):834–44. [12] Simeonovic CJ, Wilson JD. Xenotransplantation of fetal pig proislets in anti-CD4treated diabetic NOD/Lt mice. Transplant Proc 1992;24(5):2287–8. [13] Guo Z, Wu T, Kirchhof N, Mital D, Williams JW, Azuma M, et al. Immunotherapy with nondepleting anti-CD4 monoclonal antibodies but not CD28 antagonists protects islet graft in spontaneously diabetic nod mice from autoimmune destruction and allogeneic and xenogeneic graft rejection. Transplantation 2001;71(11):1656–65. [14] Koulmanda M, Qipo A, Smith RN, Auchincloss Jr H. Pig islet xenografts are resistant to autoimmune destruction by non-obese diabetic recipients after anti-CD4 treatment. Xenotransplantation 2003;10(2):178–84. [15] Appel MC, Banuelos SJ, Greiner DL, Shultz LD, Mordes JP, Rossini AA. Prolonged survival of neonatal porcine islet xenografts in mice treated with a donor-specific transfusion and anti-CD154 antibody. Transplantation 2004;77(9):1341–9. [16] Morelli AE. The immune regulatory effect of apoptotic cells and exosomes on dendritic cells: its impact on transplantation. Am J Transplant 2006;6(2):254–61. [17] Emanuel EJ, Wendler D, Grady C. What makes clinical research ethical? JAMA 2000;283(20):2701–11. [18] H.Y. Vanderpool. Informed consent and xenotransplantation clinical trials. Xenotransplantation; in press. [19] Fishman JA, Patience C. Xenotransplantation: infectious risk revisited. Am J Transplant 2004;4(9):1383–90. [20] Sykes M, d'Apice A, Sandrin M. Position paper of the Ethics Committee of the International Xenotransplantation Association. Transplantation 2004;78(8):1101–7. [21] Sykes M, Cozzi E. Xenotransplantation of pig islets into Mexican children: were the fundamental ethical requirements to proceed with such a study really met? Eur J Endocrinol 2006;154(6):921–2. [22] Kean LS, Gangappa S, Pearson TC, Larsen CP. Transplant tolerance in non-human primates: progress, current challenges and unmet needs. Am J Transplant 2006;6 (5 Pt 1):884–93. [23] U.S.Department of Health and Human Services, Food and Drug Administration, (CBER). Guidance for industry: source animal, product, preclinical and clinical issues concerning the use of xenotransplantation products in humans; 2003. Ref Type: Electronic Citation http://www.fda.gov/cber/gdins/clinxeno.htm. [24] Bennet W, Sundberg B, Lundgren T, Tibell A, Groth CG, Richards A, et al. Damage to porcine islets of Langerhans after exposure to human blood in vitro, or after intraportal transplantation to cynomologus monkeys: protective effects of sCR1 and heparin [see comments]. Transplantation 2000;69(5):711–9. [25] Buhler L, Deng S, O'Neil J, Kitamura H, Koulmanda M, Baldi A, et al. Adult porcine islet transplantation in baboons treated with conventional immunosuppression or a non-myeloablative regimen and CD154 blockade. Xenotransplantation 2002;9 (1):3–13. [26] Cantarovich D, Blancho G, Potiron N, Jugeau N, Fiche M, Chagneau C, et al. Rapid failure of pig islet transplantation in non human primates. Xenotransplantation 2002;9(1):25–35.
While the recent unprecedented accomplishments in preclinical pig-to-NHP islet cell xenotransplantation hold great promise and may have far-reaching implications, they also show important limitations. However, these limitations do not appear insurmountable. Investigators seeking to develop clinically applicable immunosuppressive protocols focus their efforts on the evaluation of immunosuppressive agents that could substitute for anti-CD154 antibodies. These antibodies are a critical component of three protocols that were effective in preventing the rejection of adult pig islets in monkeys [39,43,47]; however, the clinical development of these antibodies was stopped due to severe thromboembolic risks [61]. Preliminary results suggest that antagonistic anti-CD40 monoclonal antibodies could be equally effective when combined with IL-2R antibodies, CD28 antagonists, and sirolimus [48]. Several other immunosuppressants, both investigational and approved, are currently being investigated [62]. Ideally, and in view of the on-demand availability of xenogeneic islet tissue, immunosuppressive drugs should be selected based on their ability to facilitate negative vaccination strategies involving the pretransplant administration of donor antigen to the quiescent immune system of the recipient [63,64]. A large number of islet product-directed strategies are currently under investigation to allow minimization of immunosuppressive requirements. These include the procurement of presumably less immunogenic embryonic pancreatic precursor tissue [49,65], pretransplant islet culture in the presence of mitomycin-C to upregulate TGF-β production of islets [66], surface heparinization [67] or pegylation [68,69] of islets, transfection islets with immunorepellant SDF-1 [70], transgenic islets overexpressing complement regulatory proteins [43,71] and immunoisolation [36,45,46], to name a few. Ultimately, all these strategies share the central objective of creating an immunoprivileged microenvironment at the islet implantation site and/or draining lymph node. It seems therefore desirable to pursue these concepts as part of a multifunctional design that also integrates the local and controlled release of immunoregulatory, proangiogenic, and cytoprotective factors, separate or together with microspheres, nanoparticles, and bioscaffolds [72–75]. 7. Conclusions Building on the remarkable recent progress in the stringent preclinical pig-to-NHP model, research teams are currently pursuing several tantalizing concepts in the promising field of islet xenotransplantation. The simultaneous development of suitable source pigs will position the scientific community to translate these findings safely to the clinic to ultimately provide tangible benefits for patients and their families who are unfairly burdened by type 1 diabetes. Contributors BJH wrote the paper; NW contributed to the compilation of data summarized in Tables 1 and 2. Acknowledgments The work presented in this summary was supported in part by grants from the Juvenile Diabetes Research Foundation (JDRF PPG #21-2006-
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