[Show abstract][Hide abstract] ABSTRACT: Natural killer T cells are immunoregulatory cells, which have important roles in tolerance and autoimmunity, as demonstrated primarily in mice and humans. In this study, we define the phenotype and function of Valpha24(+) T cells derived from the spleens of rhesus macaques, a species increasingly used in models of immune tolerance. Valpha24(+) cells were isolated and expanded with monocyte-derived immature dendritic cells in the presence of alpha-galactosylceramide, IL-2, and IL-15. Rhesus NKT cells were stained with mAbs against both Valpha24 and the invariant complementarity-determining region 3 epitope of the human Valpha24/JalphaQ TCR. The cells were CD4, CD8 double negative and expressed CD56. Rhesus NKT cells also exhibited moderate to high expression of CD95, CD45RO, CD11a, and beta(7) integrin, but did not express CD45 RA, CD62L, CCR7, CD28, and other activation, costimulatory molecules (CD69 and CD40L). By intracellular staining, >90% of unstimulated rhesus NKT cells expressed IL-10, but not IFN-gamma. However, the latter was strongly expressed after stimulation. Rhesus NKT secreted large amounts of TGF-beta, IL-13, and IL-6, and modest levels of IFN-gamma, whereas IL-10 secretion was negligible and no detectable IL-4 was observed either intracellularly or in culture supernatants. Functionally, the NKT cells and their supernatants suppressed T cell proliferation in allogeneic MLR. We conclude that long-term cultured rhesus macaque spleen-derived Valpha24(+) T cells are semi-invariant double-negative cells with effector memory phenotype. These cells are semianergic, polarized to a uniquely Th3 > T regulatory-1 regulatory cell phenotype, and have regulatory/suppressive function in vitro.
The Journal of Immunology 10/2003; 171(6):2904-11. · 5.36 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We have demonstrated the presence of crystalline particles in University of Wisconsin (UW) solution used to perfuse rhesus monkey kidney transplants. These particles were visible in obstructed blood vessels and associated with immediate graft thrombosis and necrosis. This occurred in 25.7% of kidneys perfused with UW solution and transplanted into young, unsensitized recipients. Two molecular species of crystals were defined by mass spectrometry. The particle size ranged from 3 to greater than 100 microm, with a preponderance of particles less than 25 microm in diameter. Such particles are not removed by 40-microm filtration, but can be removed by centrifugation. With extensive use of UW solution for organ storage, the potential for particle-induced damage in small vessels in both experimental and human transplants needs to be carefully scrutinized.
Transplant International 09/2003; 16(8):491-6. · 3.16 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Brief treatment of rhesus macaques with immunotoxin plus 15-deoxyspergualin has yielded exceptional numbers (54%) of stable tolerant kidney allograft recipients, surviving over 6 years without rejection or immunosuppression. An early increase in IL-10 and reduction in IFNgamma distinguished recipients that subsequently became tolerant. Furthermore, analysis suggested that this immune switch was programmed within hours of transplantation. Administering deoxyspergualin within 5 h of surgery gave a higher incidence of tolerance (76%) compared to administration >5 h before or after surgery (11%, P<0.01). Deoxyspergualin inhibits nuclear translocation of activated NF-kappaB through heat shock proteins. Lymph node biopsies from tolerant recipients showed significant reductions in cytoplasmic expression of Hsp70 and RelB and almost complete inhibition of nuclear translocation of both. The early timing effect of deoxyspergualin suggests a crucial limitation to induction of stable tolerance is activation of Hsp-dependent innate responses to damage by ischemia-reperfusion. This was supported by studies in murine kidney reperfusion injury, where deoxyspergualin given 5 h before reperfusion protected renal function and reduced levels of IL-6 and IL-12. The narrow timing window for initiating deoxyspergualin treatment suggests the innate immune system is poised to defeat allograft tolerance induction, so effective blockade of NF-kappaB-mediated innate immunity must be in place early, to enable development of a tolerogenic environment.
[Show abstract][Hide abstract] ABSTRACT: Pancreatic islet transplantation (PIT) is an attractive alternative for type 1 diabetic patients. PIT is not yet an effective clinical reality due in part to early loss of functional islet mass. In addition, current immunosuppressive drugs have toxic effects on islets and increase the risk of morbidity and mortality. Precise and durable alpha- and beta-cell function is essential for the success of PIT. Therefore, it is important to establish whether PIT can produce adequate long-term metabolic control, especially in the absence of chronic immunosuppressive therapy (CIT). In the present study, the stability of functional alpha- and beta-cell mass and metabolic function was assessed in streptozotocin (STZ)-induced diabetic primates following PIT in the absence of CIT. Diabetes was induced in rhesus macaques with STZ, 140 mg/kg. Hyperglycemia was reversed rapidly by PIT coupled with a 14-day tolerance induction protocol based on F(Ab)2-IT and DSG (n = 7). Two diabetic animals received the tolerance induction protocol without PIT. Acute rejection was presented in three animals at 70, 353 and 353 days post transplant in the tolerance induction protocol, whereas the controls [F(Ab)2-IT or DSG alone] showed early 10-day function but all lost islet function by days 15-70. One recipient [F(Ab)2-IT or DSG] died euglycemic after a surgical procedure on day 187. At 2 years, three animals studied had a normal FIM evaluated by oral glucose tolerance test, mixed meal test, acute insulin response to glucose, glucose disposal rate, and hyperinsulinemic hypoglycemic clamp. PIT in STZ-induced diabetic primates resulted in restoration of normal alpha- and beta-cell function. Operational tolerance induction was achieved with only peritransplant administration of F(Ab)2-IT and DSG sparing the animals from chronic exposure of diabetogenic immunosuppressive drugs. These results offer an exciting new potential for treatment of type 1 diabetes mellitus.
American Journal of Transplantation 03/2003; 3(2):128-38. · 6.19 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The ability to transfer immunoregulatory, cytoprotective, or antiapoptotic genes into pancreatic islets (PIs) may allow enhanced post-transplantation survival. The available gene transfer vectors differ greatly in their ability to infect and express genes in different cell types. One limitation associated with the use of viral vectors is related to the virus reliance on the presence of its primary binding site. Tropism of the viral vectors can be altered using retargeting strategies. Results on phage biopanning proved that the RGD motif has in vivo targeting capabilities. This motif interacts especially with cellular integrins of the alphavbeta3 and alphavbeta5 types, highly expressed on pancreatic islets. In this report, we have explored the utility of a retargeted adenovirus vector (Ad) containing an RGD motif in the HI loop of the fiber knob in order to improve the infection efficiency to intact isolated nonhuman primate PIs and reduce toxicity after the genetic modification. Nonhuman primate Pis were isolated by a semi-automated technique. Steptozotocin-induced diabetic mice with severe combined immunodeficiency disease (SCID) were used as recipients. A recombinant Ad containing a heterologous RGD peptide and expressing luciferase (AdRGDLuc) or green fluorescent protein (AdRGDGFP) were generated in our laboratory. Similar Ads without the RGD peptide were used as a control (AdLuc and AdGFP). Higher transfection efficiency was demonstrated using AdRGDGFP compared with AdGFP (>80% of the islet cells were infected at 10 particle-forming units (pfu)/cell using AdRGDGFP vs. 7% after infection with AdGFP).More than 90% of the infected cells were insulin-producing cells. Significantly higher transgene expression was demonstrated after infection with AdRGDLuc compared with AdLuc at different titers. Analysis of the glucose-stimulated insulin response demonstrated better performance of PI transfected with AdRGDLuc at low titers (10 pfu/cell in order to achieve > 80% transfection efficiency) compared with AdLuc at high titers. Finally, long-term euglycemia (>250d) was observed in 89% of the animals that received PI infected with AdRGDLuc compared with none of the animals that received PI infected with AdLuc. The present study provides new information about the possibility of tropism modification of Ad vectors to increase the transfection efficiency and transgene expression to isolated PI. Incorporation of the RGD sequence in the HI loop of the fiber knob allows highly efficient transfection efficiency to nonhuman primate insulin-producing cells and adequate long-term function of the p-cell after transplantation.
American Journal of Transplantation 03/2002; 2(3):237-43. · 6.19 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Pancreatic islet transplantation (PIT) is an attractive alternative to insulin-dependent diabetes treatment but is not yet a clinical reality. The first few days after PIT are characterized by substantial pancreatic islet dysfunction and death. Apoptosis has been documented in PI after extracellular matrix removal, during culture time, after exposure to proinflammatory cytokines, hypoxic conditions before islet revascularization, and rejection. Targeting the apoptosis pathway by adenoviral-mediated gene transfer of the anti-apoptotic Bcl-2 gene exerts a major cytoprotective effect on isolated macaque pancreatic islets. Bcl-2 transfection ex vivo protects islets from apoptosis induced by disruption of the islet extracellular matrix during pancreatic digestion. Additionally, over-expression of Bcl-2 confers long-term, stable protection and maintenance of functional islet mass after transplantation into diabetic SCID mice. Genetic modification of PI also reduced the islet mass required to achieve stable euglycemia. Ex vivo gene transfer of anti-apoptotic genes has potential as a therapeutic approach to both minimize loss of functional islet mass post-transplant and reduce the high islet requirement currently needed for successful stable reversal of insulin-dependent diabetes [1, 2].
Kidney International 02/2002; 61(1 Suppl):S79-84. · 8.52 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: This study examines the mechanisms of early isolated islet apoptosis (II-APO) and loss of functional islet mass.
Rhesus islets were isolated for transplantation, and an aliquot was used for in vitro molecular studies of II-APO. These studies used Western blotting to examine caspase activation and perinuclear envelope protein cleavage that are associated with II-APO and used immunofluorescence analysis of Annexin V and mitochondrial permeability index to examine spontaneous and tripartite anoikis-like (TRAIL) mechanism--induced II-APO.
Caspase 6 was prominently activated in association with spontaneous II-APO, which occurred after overnight culture. In contrast, caspase 7, 8, and 9 were not activated. Cleavage of focal adhesion kinase and Lamin, substrates of caspase 6, was also evident in spontaneous II-APO. II-APO was exaggerated by the addition of the TRAIL mechanism. The TRAIL mechanism--induced II-APO was blocked by the caspase 6 inhibitor, VEID, and by the soluble fusion proteins, DR4 or DR5, which act as decoy receptors. In vivo studies in diabetic severe combined immunodeficiency disease mice showed that rhesus islets were cytoprotected by either ex vivo gene transfer of Bcl-2 or treatment of the isolated islet with VEID.
These studies suggest 3 major mechanisms involved in II-APO: caspase 6 activation, a TRAIL-induced apoptosis pathway, and the mitochondrial-associated apoptosis pathway. Inhibition of these II-APO pathways may improve isolated islet survival and reduce functional islet mass loss, which compromises the stable reversal of diabetes.
Surgery 09/2001; 130(2):333-8. · 3.11 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Isolated pancreatic islet transplantation is a promising alternative to conventional insulin-dependent diabetes treatment but is not yet a practical clinical therapy. In the first few days after pancreatic islet transplantation, substantial donor pancreatic islet dysfunction and apoptosis commonly occur. Islet apoptosis has been documented after extracellular matrix disruption and exposure to proinflammatory cytokines, and during hypoxia before islet revascularization and rejection. These studies show that targeting the apoptosis pathway by adenoviral-mediated gene transfer of the anti-apoptotic Bcl-2 gene exerts a major cytoprotective effect on isolated macaque pancreatic islets. Bcl-2 transfection ex vivo protects these islets from apoptosis induced by disruption of the islet extracellular matrix during pancreatic digestion. Additionally, overexpression of Bcl-2 confers long-term, stable protection and maintenance of functional islet mass after transplantation of macaque islets into diabetic severe combined immunodeficency mice. Notably, genetic modification of pancreatic islets also reduced the islet mass required to achieve stable euglycemia. Ex vivo gene transfer of anti-apoptotic genes has potential as a therapeutic approach to both minimize loss of functional islet mass after transplant and reduce the high donor islet requirement currently needed for successful stable reversal of insulin-dependent diabetes.
[Show abstract][Hide abstract] ABSTRACT: This review considers whether protocols for deliberate induction of transplant tolerance to organ allografts will become a clinical reality. Three current nonhuman primate (NHP) preclinical models that seem likely to lead to clinical tolerance are discussed. These are (1) donor bone marrow in combination with antibody, or immunotoxin or irradiation or both; (2) costimulatory blockade with or without donor bone marrow, and (3) immunotoxin-based T-cell depletion combined with deoxyspergualin to block early posttransplant NF-κB-dependent dendritic cell maturation and proinflammatory cytokine responses. Each model can prevent acute allograft rejection, making prevention of chronic rejection the goal for establishing clinical efficacy. It is noteworthy that the first and third strategies have yielded numerous long-term, drug-free tolerant NHP recipients (kidney and islet transplants) who are free of measurable chronic rejection. As these three NHP strategies are refined and critically replicated by collaborating centers, transplant tolerance induction will be poised for clinical application.
Current Opinion in Organ Transplantation 02/2001; 6(1):95-101. · 2.38 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Advances in immunotoxin formulation have enabled application of this reagent to tolerance induction in primate preclinical models. The most successful paradigm transiently ablates recipient T cells, enabling engraftment into an immunologically permissive environment. The greatest success in stable tolerance induction (without chronic rejection) has been with the combination of immunotoxin and 15-deoxyspergualin. 15-deoxyspergualin complements immunotoxin by reducing cytokine production and blocking dendritic cell maturation and antigen presentation. Thus, we have been able to establish durable, long-term immunologic T- and B-cell tolerance (without donor-specific alloimmunity) to MHC-mismatched macaque allografts, satisfying the most stringent criteria for true toilerance.
Abbreviations:Ab antibody, DSG 15-deoxyspergualin, DT diphtheria toxin, F(Ab)2 pepsin fragment of antibody, IT immunotoxin, mAb monoclonal antibody
Immunotoxins (ITs) are selective cytotoxic agents whose use falls under the category of targeted immunotherapy. The application of IT to a variety of therapies, particularly for cancer, generally has yielded disappointing or equivocal results. However, new formulations have overcome some of the previous pharmacological shortcomings and systemic toxicities. The improved performance of these newer reagents has attracted interest in ITs for use in transplant tolerance induction paradigms involving specific T-cell cytoablation . Our anti-CD3ϵ-IT approach to tolerance, although seemingly aggressive, originates from the concept that stable immunological tolerance in an outbred species can be induced through T-cell ablation and regeneration in the presence of a functioning allograft, all within a quiescent immunologic environment. The obvious benefits of transplantation employing immune tolerance are freedom from a lifetime of immunosuppressive drugs and the elimination of chronic rejection.
Current Opinion in Organ Transplantation 02/2000; 5(1):29-34. · 2.38 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Background. Pancreatic islet transplantation (PIT) is an attractive alternative for patients with type I diabetes mellitus. PIT is not yet an effective clinical reality due in part to the high incidence of rejection and early loss of functional islet mass. In addition, current immunosuppressive drugs have toxic effects on islets and increase the risk of morbidity and mor-tality. In the present study, the effects of PIT on gly-cemic parameters were assessed in spontaneously di-abetic primates. Methods. Five insulinopenic nonhuman primates (three Macacca fascicularis, one Ceropithecus ae-thiops, and one Macacca mulatta) were studied. All required twice-daily treatment with 4 –10 U of insulin. For immunosuppression, the animals received anti-CD3-immunotoxin (100 g/kg/initially infused 2 hr be-fore transplantation and again on day 1), cyclospor-ine (CsA) (20 mg/kg/i.v./2 hr before transplantation), cyclosporine microemulsion (Neoral) 60 mg/kg/b.i.d. on days 1 to 3 with dose adjusted by blood levels, and methylprednisolone (15 mg/kg day 0 to 3). Three recipients were given islets from a single donor (M mulatta). The islets were prepared by a semiauto-mated technique using Liberase. A mean of 13,136 islet equivalents/kg was infused into the portal vein. Two animals (M fascicularis and M mulatta) were used as a diabetic, nontransplanted control. Several metabolic parameters were evaluated. Results. All monkeys that underwent transplanta-tion experienced reversal of diabetes mellitus with normalization of all diabetic glycemic parameters. In the nontransplanted primates given the same immu-nosuppression but no PIT, diabetic metabolic param-eters were unchanged after 9 months of follow-up. In contrast, all three PIT recipients established fasting and nonfasting euglycemia within 1–2 weeks, and none required exogenous insulin after day 10. Normal intravenous glucose tolerance tests were observed at day 15, and no significant differences in the glucose disappearance rate (Kg) were observed at days 15, 45, 190, and 365 days after transplantation. The acute in-sulin response to glucose indicated no significant re-duction of functional islet mass. Conclusions. PIT in severely insulinopenic type I diabetes mellitus primates resulted in restoration of normal glycemic parameters and durable islet mass. Operational tolerance was achieved with only 4 days of drug administration, sparing the animals from chronic exposure to potentially diabetogenic immuno-suppressive drugs. These results offer an exciting new potential for type I diabetes mellitus treatment.
[Show abstract][Hide abstract] ABSTRACT: Background. Day of transplant T cell depletion with anti-CD3 immunotoxin or F(Ab)2 immunotoxin induces stable tolerance to renal allografts in rhesus monkeys given 15-deoxyspergualin (DSG), a NF-κB inhibitor that suppresses proinflammatory cytokine (PC) production. Because PC and NF-κB are involved in dendritic cell (DC) maturation, we asked if impaired DC maturation and Th2-type cytokine deviation might be related to the synergistic effect of DSG in this novel model.
Methods. Immunosuppression was initiated 4 hr before transplanting a major histocompatibility complex mismatched renal allograft. Some groups received a supplemental 5-day course of cyclosporine A or DSG or a 15-day course of DSG. Peripheral lymph nodes were sequentially examined for presence of mature DC. In vitro effects of DSG on PC-induced maturation of DC were also examined.
Results. Allografts survived without rejection in 87% of recipients given immunotoxin or F(Ab)2 immunotoxin with DSG × 15 days, in 50% with DSG × 5 days, and 0% with cyclosporine A. The longest DSG survivors are >1000 days with normal graft function and tolerance validated, including acceptance of challenge second donor kidneys without treatment. DSG-treated recipients were unique in developing polarized Th2-type plasma cytokines. In DSG recipients, mature DC were significantly reduced in day +5 lymph node biopsies, with complete repopulation by 30 days. In vitro studies verified an inhibitory effect of DSG on DC maturation.
Conclusions. The study suggests DSG arrests DC maturation. The unusual synergy of immunotoxin and DSG apparently involves coincidental reduction in lymph node T cell mass and mature DC, a transient circumstance favoring development of stable tolerance.
[Show abstract][Hide abstract] ABSTRACT: Background. Isolated pancreatic islet transplantation (IPITx) is an attractive alternative for treatment of insulin-dependent diabetes mellitus (IDDM). However, IPITx has been difficult to implement clinically because islets frequently fail to function, have a high incidence of rejection, and are susceptible to autoimmune recurrence and damage by chronic immunosuppressive therapy. Tolerance induction may be a rational approach to resolve several of these limitations. Because anti-CD3 immunotoxin (IT) has been successful in promoting stable primate kidney transplant tolerance in our experience, we considered that tolerance induction with IT might be duplicated in IPITx.
Materials and Methods. Three monkeys with spontaneous IDDM (two Macaca fascicularis and one Ceropithecus aethiops) were treated with xenogeneic pancreatic islets (Macaca mulatta). Intrahepatic islet transplantation was performed at a mean of 13136±3860 islet equivalents/kg. Islet xenograft acceptance was accomplished by tolerance induction with two injections of IT given on day 0 at 2 hr before transplantation and on day +1, respectively. IT treatment was supplemented with cyclosporine and steroids administered on days 0 through 4. No additional immunosuppression was given thereafter. Two additional control macaques with spontaneous IDDM received the immunosuppressive protocol without islet infusion.
Results. All recipients were restored to stable euglycemia, off exogenous insulin, within 1-2 weeks after transplantation. Glucose tolerance, C-peptide, and glycosylated hemoglobin tests confirmed the restoration of normal glucose homeostasis after islet transplantation. All three islet recipients have remained euglycemic at 410, 255, and 100 days of follow-up despite recovery of peripheral T cells to normal levels. In contrast, none of the controls presented changes in the diabetic status 4 and 8 months after treatment.
Conclusions. These results represent the first demonstration in nonhuman primates of stable, long-term acceptance of nonencapsulated xenogeneic islets off all immunosuppression, suggesting operational tolerance. The findings have potential implications for islet transplantation as well as improved and more cost-effective therapy for IDDM.