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ILAR Journal Vol 45(3)

Nonhuman Primate Models for Islet Transplantation in Type 1 Diabetes Research

Lakshmi K. Gaur

Lakshmi K. Gaur, Ph.D., is a Research Scientist at the Washington National Primate Research Center, Department of Microbiology, University of Washington School of Medicine, Seattle, Washington.

Abstract

Insulin-dependent diabetes mellitus is an autoimmune disease that causes a progressive destruction of the pancreatic beta cells. As a result, the patient requires exogenous insulin to maintain normal blood glucose levels. Both the pancreas and the islets of Langerhans have been transplanted successfully in humans and in animal models, resulting in full normalization of glucose homeostasis. However, insulin independence, transient or persistent, was documented in only a small fraction of cases until recently. The chronic immunosuppression required to avoid immunological rejection appears to be toxic to the islets and adds the risk of lymphoproliferative disease reported earlier. For islet transplantation to become the method of choice, it is essential first to identify islet-friendly immunosuppressive regimens and/or to develop methods that induce donor-specific tolerance and improve islet isolation and transplantation protocols. Indeed, researchers have already successfully allografted islets in the presence of nonsteroidal immunosuppression in a process known as the Edmonton protocol. An alternative method, gene therapy, could replace these other methods and better meet the insulin requirement of an individual without requiring pancreatic or islet transplantation. This alternative, however, requires animal models to develop and test clinical protocols and to demonstrate the feasibility of preclinical trials. Nonhuman primates are ideally suited to achieve these goals. The efforts toward developing a nonhuman primate diabetic model with demonstrable insulin dependence are discussed and include pancreatic and islet transplant trials to reverse the diabetic state and achieve insulin independence. Also described are the various protocols that have been tested in primates to circumvent immunosuppression by using tolerance induction strategies in lieu of immunosuppression, thus exploring the field of donor-specific tolerance that extends beyond islet transplantation.

Key Words: islet transplantation; islets; major histocompatibility complex; nonhuman primates; pancreatectomy; streptozotocin; tolerance induction

Introduction

Insulin-dependent diabetes mellitus, often referred to as type 1 diabetes and formerly known as juvenile onset diabetes, can occur in early childhood to young adulthood. The disease is an autoimmune disease, although the specific mechanisms that trigger the autoimmune destruction of the pancreatic beta cells still remain elusive (Apostolou et al. 2003; Mandrup-Poulsen 2003).

Patients with type 1 diabetes must learn to maintain their blood glucose levels close to normal by controlling their diet and by carefully monitoring their insulin intake until a permanent cure can be found for diabetes. Pancreas transplantation is the first and most established treatment option for type 1 and type 2 diabetes mellitus. The patient survival rate is 91%, and the 1-yr graft survival rate is only 71% in some populations (Kubota and Makuuchi 1996). Technical failure and chronic rejection still account for a failure rate of >20% (Humar et al. 2003).

Islet transplantation is expected to be more efficacious than pancreas transplantation, but until recently, this procedure has not met with great success. Its lack of success could be due to the loss of functional islet mass over time and/or to the earlier use of ineffectual immunosuppressive regimens. Recently modified immunosuppressive regimens appear to have improved this outcome (Shapiro et al. 2003). In spite of recent improvements, however, the success rate of islet transplantation must be increased significantly to achieve tolerance to the donor islets without immunosuppression.

Tolerance to transplantation is not a novel concept. Medawar and coworkers originally demonstrated that injection of donor bone marrow into immunoincompetent neonatal rodents could induce tolerance to grafts from animals of the same strains as the bone marrow donor strains (Billingham et al. 1953). Thus, future progress relies on the development of strategies that are alternatives to the current immunosuppressive drugs. There is a compelling need for an animal model close to the human in which to develop and test strategies to improve islet cell graft survival and alternatives to immunosuppression before human trials can take place.

Animal Models Tested for Diabetes

Diabetes has been described in various animals, from rodents (Makino et al. 1980; Nakhooda et al. 1976) to nonhuman primates (Gaur et al. 2001). Although Leblanc documented diabetes in a monkey as early as 1851 (LeBlanc 1851), the disease in macaques has not been well characterized. Nevertheless, the primate pancreas has been used most commonly in the indirect immunofluorescence test for islet cell antibodies (Nozaffarian et al. 1991), which reveals a close phylogenetic relationship. Macaca nemestrina islets were shown to express the islet GAD65 autoantigen (Hagopian et al. 1993), which has been a useful source to screen for novel autoantigens using sera from new onset insulin-dependent diabetes mellitus patients.

Immunological responses in macaques are very similar to those in the human. The responses can be analyzed readily using many of the same antibody reagents and nucleotide probes used in human, but not in rodent, studies. Analyses beginning in the 1980s have provided ample evidence that the major histocompatibility complex (MHC¹) is highly conserved through primate evolution both at serologic and molecular levels (Gaur and Nepom 1996; Gaur et al. 1988, 1992; Gyllensten and Erlich 1989; Mayer et al. 1988). Thus, nonhuman primates represent important experimental animal models for studies of biomedical research and, in particular, for immunologic studies of autoimmunity in which there is a relationship between the MHC class II and susceptibility.

In clinical research, macaques and baboons have been used extensively as transplantation models (Gengozian et al. 1966; Rabson et al. 1965; Thomas and Epstein 1965; Willman et al. 1965), as disease models (Bingaman and Bakay 2000; Greep 1970; Pitkin and Reynolds 1970), and in therapeutic trails (Whitcomb et al. 1966). At about the same time, Hans Balner recognized the need to develop reagents to define MHC in animals ranging from rhesus macaques (Macaca mulatta) to chimpanzees (Pan troglodytes) to facilitate the role of nonhuman primate models in biomedical research (e.g., Balner 1974, 1981; Balner and Van Rood 1971; Balner et al. 1967). Among the nonhuman primates, baboons (Papio) and macaques (Macaca) have been widely utilized. The potential of nonhuman primate models in diabetes research has been well understood, and numerous investigators from the early 1970s to very recently have used nonhuman diabetes models (Gaur et al. 2001; Howard 1982; Koulmanda et al. 2003; Mintz et al. 1972). Whole pancreatic or islet transplant trials in these models have utilized a variety of immunosuppressive regimens (Gaur et al. 2002a,b; Kawai et al. 2001; Kenyon et al. 1999b; Levisetti et al. 1997; Scharp et al. 1975; Thomas et al. 2001a).

Induction of Beta-Cell Destruction Leading to Diabetes

Because very few spontaneous diabetic models exist (Clarkson et al. 1985; Howard and Palotay 1975), diabetes has been induced for controlled experiments in nonhuman primates with streptozotocin (STZ¹), alloxan, hypothalamic lesions, or pancreatectomy (Howard 1982). Pancreatectomy with or without STZ has been used successfully to render insulin deficiency in animal models (Ericzon et al. 1991; Gray et al. 1986; Hirshberg et al. 2002; Kenyon et al. 1999a).

Use of STZ

STZ has been used extensively to induce diabetes in experimental animals because it is the least invasive and perhaps the most efficient way to induce diabetes. In mice, STZ has induced an autoimmune type of disease (Koevary et al. 1983; Like et al. 1985; Whalen et al. 1994). Insulitis and diabetes can be induced in certain strains of inbred mice by the injection of multiple subdiabetogenic doses of STZ (Leiter 1982; Rossini et al. 1977). This induced diabetes can be prevented by treatment with a crude preparation of antilymphocyte globulin (Rossini et al. 1977) or monoclonal antibodies against Ia antigens (Kiesel and Kolb 1983). The development of STZ-induced diabetes is prevented by total body irradiation, but diabetes develops after adoptive replacement of T-lymphocytes (Nedergaard et al. 1983; Rossini et al. 1978). Previous studies (e.g., Harold et al. 1987) support the hypothesis that an immune response is important to the development of multi-low-dose STZ diabetes and indicate that treatment with monoclonal antibodies against the L3T4+ or Lyt2+ T-lymphocyte subsets can attenuate this process. The resistance of nude mice and thymectomized mice to STZ (Paik et al. 1980) and the results of adoptive transfer experiments (Buschard and Rygaard 1977) and reconstitution experiments (Paik et al. 1982; Paik et al. 1980) all support a role of T cells in the development of diabetes after low-dose STZ. In an effort to recapitulate this model in macaques, several investigators (Gaur et al. 2001; McCulloch et al. 1991; Takimoto et al. 1988) have used single to multiple low-dose STZ treatment in macaques and baboons to achieve beta-cell destruction.

Species-specific Characteristic of STZ

It is important to establish the dose of STZ that is required to induce irreversible diabetes with complete beta-cell loss and insulin dependence before defining treatment strategies. Numerous doses have been reported to have induced diabetes in various nonhuman primate species, and it is apparent from these studies that a particular dose in one species may either lack the effect in other species (e.g., Old World monkeys) or may indeed be toxic at slightly higher doses. Multiple low doses of 40 mg/kg were reported to induce efficient beta-cell destruction in mice (Leiter 1982) but did not have the same result in M. nemestrina (Gaur et al. 2001). Such multiple doses, as well as a single high dose (150 mg/kg), resulted in fatal kidney failure in M. nemestrina. (Gaur et al. 2001; Gaur unpublished observations).

It is interesting to note that animals carrying certain MHC class II genes of the DQA1*03 lineage have developed beta-cell damage at an accelerated pace (Gaur et al. 2001). A single dose of STZ induces beta-cell destruction in various species of macaques (Figure 1); however, the doses vary from 40 mg/kg (Gaur et al. 2001) to 150 mg/kg (Theriault et al. 1999). A single low dose of STZ administered to baboons was not sufficient (McCulloch et al. 1991) because follow-up detected a transient drop in beta-cell function, although further destruction of beta cells was not observed. In fact, 8 wk after STZ treatment, all beta-cell function tests, except glucose potentiation, returned to pre-STZ levels. When high doses of STZ were used, hyperglycemia resulted shortly thereafter (Weigle et al. 1991). Therefore, the resulting beta-cell destruction was secondary to the toxic effect of the drug. It is unknown whether macrophage infiltration occurred, similar to that observed in rat islets after STZ exposure (In't Veld and Pipeleers 1988). This infiltration may be necessary for antigen processing. Although both high and low doses of STZ result in hyperglycemia and beta-cell destruction, toxic side effects due to STZ are minimal (e.g., cause beta-cell destruction without affecting kidney or liver functions) when used in low doses.

Figure 1 (A) Intravenous glucose tolerance test for a diabetic macaque before and after treatment with stretozotocin (STZ). This pigtail macaque received one 40 mg/kg dose of STZ intravenously, and 24 hr later became hyperglycemic. Before STZ treatment, fasting blood glucose for this animal was approximately 65 mg/dL. (B) Histology of two pancreata from a normal control macaque and a diabetic (right panel) macaque stained for insulin (blue) and glucagon (brown). The islets from the diabetic macaque reveal almost complete damage of insulin-secreting cells by the absence of insulin staining in the pancreas, with positive staining for glucagon. The diabetic pancreas section is from the same animal shown in A.

Nonhuman Primates as Islet Transplant Models

The islet transplant procedure, which could potentially be performed on an outpatient basis, is an attractive alternative to pancreatic transplant (Kenyon et al. 1996). If islet transplantation could be achieved with no or minimal immunosuppression, the procedure would be especially suitable for many more patients than are currently able to qualify as surgical candidates. Furthermore, islets could be obtained from those organ donors whose pancreata are currently deemed unusable due to the lack of adequate vascular conduits, thus increasing the total pool of organs and tissues available for transplant.

Clinical trials for islet transplantation are not consistent. Even before the current, still ongoing Immune Tolerance Network trials, clinical trials of islet transplantation had provided evidence of long-term (>5 yr) islet graft survival. However, these results were observed in only a fraction of the transplant recipients (Masetti et al. 1997), and a majority of islet allograft recipients either never became insulin independent or, having attained that status, eventually required a return to insulin therapy (Ricordi et al. 1992; Warnock et al. 1991). Rejection, recurrence of autoimmunity, insufficient β-cell mass, and/or problems related to islet engraftment at a heterotopic site all have been postulated to play a role in graft failure (Kenyon et al. 1996). In addition, the increased metabolic demand imposed by the use of diabetogenic immunosuppressive drugs on the transplanted islets have been clearly established (Masetti et al. 1997).

Thus, diabetogenic immunosuppressants must be eliminated to allow optimal function of engrafted islets. The immunosuppressants such as mycophenolate mofetil and rapamycin are expected to fulfill this potential (Sutherland et al. 1996). Shapiro and colleagues (2000) were successful in consecutively treating seven diabetic patients with triple noncorticoid immunosuppressive regimen in conjunction with islet transplantation. The treatment resulted in the patients' sustained freedom from the need for exogenous insulin. Initially, prolonged insulin independence was achieved in selected individuals with type 1 diabetes. This encouraging islet transplant success may increase significantly if tolerance to the donor islets can be achieved without immunosuppression.

Donor-specific Tolerance

Transplantation tolerance, defined as allograft acceptance by an immunocompetent recipient in the absence of long-term immunosuppression, has remained an elusive goal in clinical transplantation. Many tolerance protocols have been tested in animal models with variable degrees of success (e.g., Allen et al. 1997; Kenyon et al. 1999a,b). The vast majority of the strategies involving tolerance protocols are aimed at tolerizing T cells. The protocols range from bone marrow transplantation with total body irradiation to select stem cell transplantations in the absence of myeloablation. Blocking T-cell co-stimulatory signals has demonstrated extraordinary promise in some models. For short-term success, the use of islet-friendly noncorticosteroid immunosuppression has also been explored (Gaur et al. 2002b; Hirshberg et al. 2002).

Total Body Irradiation Followed by Bone Marrow Transplantation

Thomas and colleagues (1992) suggest that total body irradiation could promote allograft tolerance without chronic drug therapy. The concept of using total lymphoid irradiation (TLI¹) for immunosuppression is based on the prolonged and profound immunosuppressive effects observed after TLI in the treatment of patients with Hodgkin's disease (Du Toit and Heydenrych 1987).

Prolonged kidney and liver allograft survival was produced in baboons by low cumulative doses (500-1200 rad) of TLI (Myburgh et al. 1984). Similarly, Du Toit and Heydenrych (1987) suggest that functioning segmental pancreatic allografts are insufficient to promote normal or near normal islet function even after immunosuppression with total lymphoid irradiation. Numerous studies report allograft tolerance resulting from TLI followed by donor marrow injection in rodents as well as in nonhuman primates (e.g., Bieber et al. 1979; Carver et al. 1991). Indeed, the effectiveness of TLI in conjunction with islet allografts was reported in the 1980s (Du Toit et al. 1988; Nash et al. 1981). However, the practical application of this procedure still is suspect, due to inconsistent results and the necessity for immunosuppressive agents even after radiation treatment. For example, it appears that combined rabbit antithymocyte globulin (TLI-RATG) therapy may be beneficial in the management of transplant recipients; however, its use will probably not abolish these patients' requirements for immunosuppressive maintenance measures (Bieber et al. 1979; Carver et al. 1991).

In a baboon pancreatic allograft model, total body irradiation with cyclosporin presented with only modest pancreatic allograft survival (Du Toit et al. 1985). Nevertheless, allogeneic adult islets survived more than 100 days with TLI and donor bone marrow (with established chimerism before islet allografting) across major rat histocompatibility barriers (Britt et al. 1982). However, the clinical applicability of TLI-induced immunosuppression is a challenge in light of conflicting observations. Although Levite and Reisner (1993) observed "induction of prolonged tolerance to third-party skin graft following fully allogeneic bone marrow transplantation in mice," Papalois and colleagues (1995) observed "indefinite acceptance of heart but not skin or islet allografts in rats by total lymphoid irradiation without intrathymic injection of donor cells."

Even the previously published models of intrathymic tolerance in rodents have utilized either host myeloablation (Lorenz and Allen 1989; Matsuhashi et al. 1991) or depletion of mature host T cells by antilymphocyte globulin (Nakafusa et al. 1993; Odorico et al. 1993). Growing evidence suggests that suppressive or regulatory T cells may play a significant role in both peripheral (Thomas et al. 1994) and intrathymic (Nakafusa et al. 1993) maintenance of tolerance in an environment that contains mature and potentially active cytotoxic host T cells. If the suggestion is accurate, the question arises as to whether antilymphocyte globulin is, in fact, a necessity (Nakafusa et al. 1993) or whether its use might, instead, inhibit (Wren et al. 1992) the development of suppressor or "veto" cells. In the Pittsburgh experience, it was frequently those patients who were noncompliant in taking their immunosuppressive medications who had the greatest incidence of microchimerism (Starzl et al. 1992a).

Immunonodulatory Properties of Ultraviolet (UV¹) Treatment

Studies in animal models report the identification of the immunomodulatory properties of UV-B light that are beneficial for allograft survival (Benhamou et al. 1995). The culture of mouse pancreatic islets in an oxygen-rich atmosphere before transplantation facilitates long-term allograft survival without the use of immunosuppression. A comparison of the capacity of UV-irradiated and live spleen cells of donor origin to induce allograft rejection revealed that UV-irradiated spleen cells were not immunogenic. Live spleen cells were immunogenic, and their injection triggered allograft rejection (Agostino et al. 1983). This difference may be due to T cell anergy, but not to T cell clonal deletion.

Co-stimulation Blockade and Transplantation

Co-stimulation blockade is essentially the blockade of T cell functions. T cell activation is the result of antigen-specific interactions with the TCR/CD3 complex and co-stimulation via other T cell surface receptors. Prevention of co-stimulation can result in clonal anergy (Baliga et al. 1994). Co-stimulatory blockade has been shown to prolong allograft survival in different transplant models (Adams et al. 2002; Birsan et al. 2003; Kirk et al. 1999).

Blockade of CD40-CD40 Ligand

The co-stimulatory signal is delivered through several molecules expressed on T cells (CD28, CD152, and CD154) and antigen-presenting cells (CD80, CD86, CD40). A large body of data is available concerning various co-stimulation blockade schemes to induce specific tolerance (Kenyon et al. 1999a,b; Koenen and Joosten, 2000; Zheng et al. 1999). Blockade of the CD40-CD40 ligand pathway was expected to potentiate the capacity of donor-derived dendritic cell (DC¹) progenitors to induce long-term allograft tolerance (Lu et al. 1997). When the dendritic cell progenitors lacking surface expression of co-stimulatory molecules CD80 and CD86 were administered systemically before transplantation, Rastellini and colleagues (1995) observed prolonged survival of pancreatic islet allograft.

Kenyon and collaborators (1999a,b) have successfully prolonged islet allografts while maintaining normoglycemia in two different genera of nonhuman primates without immunosuppression. Animals were pancreatectomized to induce insulin dependency and then received intrahepatic islet allografts (10,000-40,000 islet equivalents/kg). The tolerance induction therapy consisted of 20 mg/kg of anti-CD154 or hu5c8 (Biogen) on postoperative days -1, 0, 3, 10, and 18. Maintenance therapy at 10 to 20 mg/kg was initiated on day 28 and continued thereafter once every 4 wk. The islet allografts in the monkeys receiving tolerance induction therapy were maintained for long periods of time and remained normoglycemic, whereas untreated control monkeys rapidly rejected the islet allografts. Treatment with monoclonal antibodies against CD154 was shown to prevent acute rejection in rhesus monkey recipients of kidney and skin allograft transplants for prolonged periods (Haanstra et al. 2003; Kirk et al. 1999).

There is insufficient evidence in the literature to declare that anti-CD40L induces permanent transplant tolerance (Haanstra et al. 2003; Kirk et al. 1999), because all monkeys have developed donor-specific immunoglobulin (Ig¹)G, and their grafts have remained infiltrated by inflammatory cells. Recently, Birsan and colleagues (2003) also reported that sirolimus (rapamycin) could be successfully combined with humanized anti-CD80 and anti-CD86 monoclonal antibodies (Birsan et al. 2003), although a significant improvement over the induction therapy by itself has not been reported to date.

B7-CD28 and B7-CD152 Blockade

Although B7-CD28 interaction results in activation, B7-CD152 interaction has an inhibitory effect on T-cell activation (Walunas et al. 1996). CTLA4-Ig is a recombinant fusion protein that consists of the extracellular domain of CD152 linked to the constant region of IgG1 and that binds both to CD80 and to CD86 simultaneously. Treatment with CTLA4-Ig had remarkable results in rodents, but only modestly prolonged allograft survival in nonhuman primates (Kirk et al. 1997; Levisetti et al. 1997).

Anti-CD3-immunotoxin and Allograft Tolerance

Thomas and coworkers have explored the use of anti-CD3 immunotoxin, which essentially induces immunological tolerance through selective ablation of T cells (Meng et al. 1998) of both naive and memory phenotypes (Hubbard et al. 2001; Thomas et al. 1997). 15-Deoxyspergualin concomitantly blocks proinflammatory cytokine production and the maturation of dendritic cells by inhibiting nuclear factor-κB transport into the nucleus (Contreras et al. 1998; Thomas et al. 1999). For years, the investigators have successfully maintained rhesus kidney allografts in the absence of immunosuppression under the anti-CD3 diphtheria-based immunotoxin tolerance cover. Four of seven STZ-diabetic rhesus macaques receiving islets from MHC-mismatched single donors under the tolerance regimen described above demonstrated prolonged insulin-free graft survival for more than 1 yr without maintenance of immunosuppressive therapy (Thomas et al. 2001b). This protocol shows great promise (see also Contreras et al. 2004, in this issue).

Donor Stem Cell Transplants Without Myeloablation to Induce Tolerance

The finding of low frequencies of circulating donor cells, microchimerism, in the peripheral blood of liver transplant recipients who were free from rejection suggested that hematopoietic microchimerism might correlate with tolerance to solid organ allografts (Starzl et al. 1992b). This finding was later confirmed in 21 such liver recipients who have now been completely weaned off immunosuppression (Mazariegos et al. 1995). The corollary hypothesis, that the induction of microchimerism might produce a state of tolerance, is now under intense investigation. In both experimental and clinical work to date, achieving stable hematopoietic chimerism by peripheral marrow infusion appears to require host myeloablation, as an extreme measure (Moses et al. 1989; Sharabi et al. 1992), and host T cell depletion, at the least (Thomas et al. 1994). In a previous primate trial, induction of chimerism by total body radiation and subsequent transplantation of T cell-depleted autologous marrow resulted in survival of only 25% of recipients at 3 mo (Wren et al. 1992). Even with toxic irradiation coupled with bone marrow, which led to a chimeric state, the graft outcome was not consistent (Kimikawa et al. 1997). Clearly, less toxic means of producing chimerism warrant exploration.

Possibility of Inducing Tolerance via Microchimerism

The number and lineage of passenger leukocytes contained in solid organ grafts vary considerably among different organs. Starzl and colleagues (1992a) postulated that the acceptance of whole organ transplantation is associated with a state of long-term mixed allogeneic or xenogeneic chimerism after the migration from the graft and widespread seeding in the recipient of dendritic and other immune cells.

Various cell populations have been implicated in facilitating allograft tolerance in a donor-specific manner, from T cell subsets (Exner et al. 1997; Neipp et al. 1999) to dendritic cells (Rastellini et al. 1995). Using whole marrow for the inoculum and introducing mature, immunocompetent donor T cells into an immunosuppressed host poses the risk of graft versus host disease (Perico et al. 1992). The use of purified, T and B lymphocyte-depleted CD34+ marrow fractions for allogeneic bone marrow transplantation in baboons resulted in engraftment without graft versus host disease (Andrews et al. 1992). These results form one rationale for pursuing the CD34+ cells for solid organ graft tolerance. Selected CD34+ fractions injected into thymus resulted in persistent microchimerism, possibly leading to donor-specific tolerance in baboons (Allen et al. 1997; Nitta et al. 2001a,b). This protocol was later extended to the pigtail macaque islet allograft model and replacing intrathymic implantation with peripheral infusion of stem cells (Gaur et al. 2002a).

Irreversible beta-cell destruction resulting in insulin dependency was achieved with a single low-dose (40-mg/kg) STZ treatment in M. nemestrina and with multiple low-dose STZ injections in a baboon (Gaur et al. 2001). Islets (10,000-30,000 IEQs/kg) from MHC-incompatible single donors were transplanted by portal infusions (Gaur et al. 2002a,b). The insulin-dependent diabetic pigtail macaques (M. nemestrina) and baboon (Papio cynocephalus anubis) received islet allografts from MHC-disparate donors in conjunction with peripheral injections of stem cell fractions, with no immunosuppression. The control animals received islet allografts alone, without immunosuppression. Five of seven macaques and the baboon that received stem cell infusions at the time of islet allografts have shown allograft survival longer than the group of macaques that received islets without the stem cell infusion. One of these five macaques remained normoglycemic for more than 3 yr, with no exogenous insulin. This macaque received stem cell populations enriched for CD34+ cells with depletion of CD18 cells, which have shown low or no allostimulatory potential in mixed lymphocyte cultures (Gaur et al. 2002a). Increased levels in insulin and C-peptide were shown in the macaques after islet transplantation. The baboon was normoglycemic for 3 mo before euthanasia was performed to detect transplanted islets in the liver. The histology of its pancreas revealed almost complete damage of insulin-secreting cells in pancreas, whereas the insulin-secreting beta cells were found in the liver. However, these studies were severely hampered by islet isolation methods, which have now been resolved (Matsumoto et al. 2003).

Use of Islet-friendly Immunosuppression

Although the search for an efficacious and feasible tolerance induction protocol continues, it is also important to continue to explore islet-friendly immunosuppression to fulfill the short-term necessity of prolonging allograft survival. The recent clinical trials in humans with noncorticosteroidal immunosuppressive regimens (Shapiro et al. 2000) and a nonhuman primate trial using CD154 co-stimulation blockade (Kenyon et al. 1999a) have catalyzed new approaches and new opportunities. Seeking reproducibility of their data, the National Institute of Allergy and Infectious Diseases proposed to repeat the Edmonton protocol in islet transplant centers through the Immune Tolerance Network. Currently, the following nine centers are participating clinical sites: (1) University of Alberta Islet Transplantation Program, Edmonton, Alberta, Canada; (2) Diabetes Research Institute, University of Miami, Miami, Florida; (3) Diabetes Institute for Immunology and Transplantation, University of Minnesota, Minneapolis, Minnesota; (4) Juvenile Diabetes Foundation Center for Islet Transplantation, Harvard Medical School, Boston, Massachusetts; (5) Diabetes Research Training Center, Washington University, St. Louis, Missouri; (6) Pacific Northwest Research Institute, Seattle, Washington; (7) Islet Transplant Centre, Justis-Liebig University, Giessen, Germany; (8) MD, Universita' Vita Salute San Raffaele, Milan, Italy; and (9) University Hospital, Geneva, Switzerland.

The ongoing clinical trials listed above are showing great promise to date (Shapiro et al. 2003). However, the nonhuman primate model will provide the best surrogate study for these human trials. A similar observation was made in a macaque islet transplant study in our laboratory. We noticed that animals that rejected their first islet allografts had prolonged graft survival when tapering doses of mycophenolate mofetil (starting with 20 mg/kg BID to 5 mg/kg SID after 2 wk) were given at the time of the second islet transplantation (Gaur et al. 2002b). In a subsequent study, pancreatectomized rhesus receiving intrahepatic islets transplanted using the Edmonton protocol immunosuppression (Shapiro et al. 2000) remained insulin independent, although the transplanted islets were not vascularized shortly after transplant (Hirshberg et al. 2002). However, variations exist in studies with respect to the nonsteroidal immunosuppressive regimens, the combinations of drugs used, and the doses (Gaur et al. 2002b; Morris 1993; Qi et al. 2000).

Summary

Despite the autotransplant success in reversing pancreatectomy-induced diabetes, similar outcomes are rare after islet cell allotransplantation (Rastellini et al. 1997; Ricordi et al. 1992). Graft failure within 3 mo after human islet allografting (Wang and Rosenberg 1999) occurred in 50% of islet transplant recipients worldwide between 1990 and 1996 (Bretzel 1996). Numerous factors have impeded the success of islet allograft transplantation, including islet mass and islet functionality. With intensified efforts in both protocols for tolerance induction and islet isolation, islet transplantation is very close to becoming a routine procedure in the clinical setting. Co-stimulation blockade with bone marrow (Durham et al. 2000) or without bone marrow (Kenyon et al. 1999a,b; Kirk et al. 1999) and methods using F(Ab)2-IT with 15-deoxyspergualin treatment show great potential.

The stem cell transplantation before or during islet transplantation is expected to abrogate the need for immunosuppression, thus eliminating the side effects due to immunosuppression in the transplant recipient. In a baboon cardiac allograft, Allen and coworkers (1997) used hematopoietic stem cells depleted of differentiation markers, which significantly reduced the allostimulatory potential of donor cells. The stem cell population enriched for CD34+ cells with depletion of CD18 cells have shown low or no allostimulatory potential in mixed lymphocyte cultures (Gaur et al. 2002a). If lack of alloimmune stimulation correlates with a propensity to tolerance induction, mixed lymphocyte reaction assays will provide an in vitro assay for the potential efficacy of various CD34+ subfractions as tolerogens.

Thus, the induction of donor-specific tolerance by either selected donor marrow populations or by co-stimulation blockade in recipients prolongs the survival of islet allografts. Further exploration and standardization of this procedure will increase our knowledge and ultimately benefit the clinical treatment diabetes.

¹Abbreviations used in this article: DC, dendritic cell; Ig, immunoglobulin; MHC, major histocompatibility complex; STZ, streptozotocin; TLI, total lymphoid irradiation; UV, ultraviolet.

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