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.

BACKGROUND:Non-human primates (NHPs) are important preclinical models for pancreatic islet transplantation (PIT) because of their close phylogenetic and immunological relationship with humans. However, low availability of NHP tissue, long learning curves and prohibitive expenses constrain the consistency of isolated NHP islets for PIT studies. To advance preclinical studies, we attempted to identify key variables that consistently influence the quantity and quality of NHP islets.METHODS:Seventy-two consecutive pancreatic islet isolations from rhesus macaques were reviewed retrospectively. A scaled down, semi-automated islet isolation method was used, and monkeys with streptozotocin-induced diabetes, weighing 3-7 kg, served as recipients for allotransplantation. We analysed the effects of 22 independent variables grouped as donor factors, surgical factors and isolation technique factors. Islet yields, success of isolation and transplantation results were used as quantitative and qualitative outcomes.RESULTS:In the multivariate analysis, variables that significantly affected islet yield were the type of monkey, pancreas preservation, enzyme lot and volume of enzyme delivered. The variables associated with successful isolation were the enzyme lot and volume delivered. The transplant result was correlated with pancreas preservation, enzyme lot, endotoxin levels and COBE collection method.CONCLUSIONS:Islet quantity and quality are highly variable between isolations. The data reviewed suggest that future NHP isolations should use bilayer preservation, infuse more than 80 ml of Liberase into the pancreas, collect non-fractioned tissue from the COBE, and strictly monitor for infection.

Background Natural killer T cells (NKT) possess dual functions of innate and adaptive immune systems, controlling viral infections and regulating autoimmune diseases. Non-human primates (NHP) are penultimate models for advancing therapeutic immunoregulatory strategies for translational application in humans, though, little is known about NHP NKT cells. Here we characterized rhesus macaque NKT cells ex vivo.Methods The frequency, phenotype and intracellular cytokine production of V alpha 24(+)6B11(+) invariant NKT (iNKT) cells were analyzed by multi-color flow cytometry. V alpha 24J alpha Q mRNA expression was analyzed by real-time RT-PCR.Results The frequencies of peripheral blood (PB) and spleen V alpha 24(+)6B11(+) iNKT cells were not significantly different. The iNKT cell subset in spleen was significantly increased for CD4(+)CD8(+) and CD3(+)CD56(+) co-expression as well as intracellular interleukin-4 production, which was rarely observed in circulating PB.Conclusion Spleen iNKT cells in rhesus macaques are Th2 biased and display phenotypically and functionally distinct profiles from their PB counterpart.

Although contemporary immunosuppressive regimens are responsible for major improvements in allograft acceptance, there are indications that long-term survival may be compromised through drug toxicity and/or chronic immune deficiency. The ultimate goal for transplantation is tolerance, defined as durable, donor-specific allograft acceptance in the absence of long-term immunosuppression. This article reviews the non human primante STEALTH model of tolerance recently developed by the transplant immunobiology group at University of Alabama at Birmingham. The STEALTH model was designed for future application to human transplantation and comprises a concise peritransplant treatment strategy of only 2 wk. Tolerance is induced by depletion of T cells, with concomitant inhibition of nuclear factor-KB/RelB-dependent proinflammatory signaling. This treatment has resulted in an unprecedented frequency of kidney allograft survival (62.5% at 3 yr), with some primate recipients remaining in good health more than 6 yr posttransplant, in the complete absence of chronic pharmacologic immunosuppression.

BACKGROUND:Tolerance is gaining momentum as an approach to reduce lifelong immunosuppressive therapy while improving transplant longevity. Anti-CD3 immunotoxin (IT), FN18-CRM9, has potential to induce tolerance owing to its exceptional ability to deplete sessile lymph node T cells. However, if initiated at the time of transplantation, alpha-CD3-IT alone elicits a proinflammatory cytokine response, precluding establishment of tolerance.METHODS:Four groups of rhesus monkeys received kidney allografts and immunosuppression. Three groups received alpha-CD3-IT alone or alpha-CD3-IT supplemented with 15-deoxyspergualin (DSG) and/or methylprednisolone (MP). One group received alpha-CD3-monoclonal antibody with DSG and MP. Cytokines were measured by enzyme-linked immunosorbent assay.RESULTS:Supplementing peritransplant alpha-CD3-IT treatment with a brief course of DSG and MP promoted rejection-free kidney allograft acceptance in 75% of macaques followed for up to 550 days. Among those given alpha-CD3-IT alone or with MP, none were long-term survivors. Tolerance developed after alpha-CD3-IT, DSG, and MP treatment, but not when the unconjugated a-CD3 monoclonal antibody was substituted for IT. Systemic production of proinflammatory cytokines interferon-gamma (IFN-gamma) and tumor necrosis factor-alpha induced after peritransplant alpha-CD3-IT was prevented only in animals given DSG. Despite high levels of interleukin (IL)-12 in the first month after transplant, tolerant recipients exhibited IL-12 resistance, as evidenced by baseline plasma levels of IFN-gamma but elevated IL-4. DSG was shown to inhibit IL-12-driven IFN-gamma production by a mechanism associated with inhibition of nuclear factor kappa-B.CONCLUSIONS:In this model, peritransplant induction of tolerance is promoted by efficient elimination of sessile lymph node T cells and control of the proinflammatory IFN-gamma response by a mechanism that appears to involve resistance to IL-12.

Department of Surgery, University of Alabama at Birmingham, Alabama, USA Correspondence to Judith Thomas, Department of Surgery, University of Alabama at Birmingham, 1670 University Boulevard, VH L107, Birmingham, AL 35294-0019, USA Tel: +1 205 934 7725; e-mail: [email protected]

The mechanisms mediating T-cell depletion plus 15-deoxyspergualin (DSG)-induced prolonged allograft survival or tolerance are uncertain. The purpose of this study is to evaluate the role of IL-4 and IL-10 in prolonged allograft survival induced by T-cell depletion plus DSG. MHC mismatched skin allograft transplantation was performed, using wild-type and three separate knockout (i.e., IL-4–/–, Stat6–/-, or IL-10–/–) mice as recipients. Induction therapy consisted of T-cell depletion and or brief course of DSG. The data demonstrate that monotherapy with T-cell-depleting mAbs or DSG prolonged skin allograft survival, compared to controls, in wild-type Balb/c recipients [median survival time (MST) = 25 and 21 vs. 10 days, p < 0.007]. T-cell depletion plus DSG further augmented skin allograft survival in wild-type animals relative to monotherapy (MST = 35 days vs. 25 and 21 days, p < 0.006 vs. mAbs or DSG only), and was equally effective in IL-4–/– and Stat6–/– recipients. In contrast, combined therapy was no better than monotherapy in IL-10–/– animals (p > 0.05). Furthermore, skin allograft survival after combined therapy was shorter in IL-10–/– versus wild-type recipients (MST 20 and 41 days, respectively, p < 0.001). IL-4-mediated signaling through Stat6 is dispensable for prolonged allograft survival induced by T-cell depletion plus DSG. In contrast, IL-10 appears to be important for prolonged allograft survival induced by combined therapy in this model.

To the Editor: We are grateful to Hubert et al. [1] for their comment on our recent publication in Diabetologia [2]. Much effort has been made to identify factors or variables that improve pancreatic islet yield and promote successful islet isolation [3, 4]. Adequate in vivo functional islet mass in the pancreas donor is one of many factors that can have an impact on the outcome of pancreatic islet isolation. Therefore, we agree with Hubert et al. [1] that a reliable method to screen functional donor islet mass in vivo has the potential to improve successful islet isolation outcomes. Other tests, such as glucose tolerance tests, beta cell secretory capacity [5], positron-emission tomography [6], and glucoseor arginineinduced acute insulin release (AIR), have been used to assess functional islet mass. Of these, arginine-induced AIR (AIRarg) is a logical choice for this purpose because it is simple and non-invasive. AIR has been used to measure functional beta cell mass in normal individuals as well as in pancreas/ islet transplant recipients [7]. The preliminary data presented by Hubert et al. demonstrating that AIRarg permitted prediction of successful islet isolation are encouraging [8, 9]. However, we caution that further testing in humans is necessary because the reported data were derived from a relatively limited number of subjects (n=29). As far as we know, there is no published standardised procedure for the AIRarg test that correlates AIRarg with functional beta cell mass for nonhuman primates (NHP). Clearly it will be useful to establish a standard AIRarg test for NHP to evaluate whether the test can be used to predict successful islet isolation in these species. Since a standardised test will allow results from different laboratories to be compared, the AIRarg test has the potential to predict successful islet isolation and functional transplantable islet mass. Pancreatic islet transplantation remains a motivating challenge for scientists and a hopeful dream for diabetic patients. Rather than a useless sacrifice of NHP, these valuable animals have given us the wonderful opportunity to understand further the complexity of pancreatic islet isolation and transplantation. A single variable might have a significant impact on the final result, but when weighed in conjunction with all the other variables involved, its effect may change dramatically. This is clearly demonstrated in the univariate and multivariate analysis performed in our study [2]. We will have to wait for further improvements in donor selection, islet isolation and implantation in order to have a reliable procedure to offer to our patients.

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-kappaB inhibitor that suppresses proinflammatory cytokine (PC) production. Because PC and NF-kappaB 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 x 15 days, in 50% with DSG x 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.

T-cell depleting anti-CD3 immunotoxins have utility in non-human primate models of transplantation tolerance and autoimmune disease therapy. We recently reported that an affinity matured single-chain (scFv) anti-monkey CD3 antibody, C207, had increased binding to T-cells and increased bioactivity in a diphtheria toxin (DT)-based biscFv immunotoxin compared with the parental antibody, FN18. However, FN18 scFvs and their mutant derivatives such as C207 did not exhibit robust bivalent character in the biscFv format. We now report that C207 in a diabody format exhibits a 7-fold increase in binding to T-cells over scFv (C207) indicating considerable divalent character for the diabody. This construct was formed by reducing the V-L/V-H linker to five residues and was secreted from Pichia pastoris as the non-covalent dimer. An immunotoxin based on this diabody format was secreted as a non-covalent dimer but was devoid of bioactivity and failed to bind T-cells, suggesting steric hindrance from the two large closely positioned truncated DT moieties. We constructed a single-chain diabody immunotoxin by fusing to the truncated DT C-terminus L-1-V-L-L-1-V-H-L-2-V-L-L-1-V-H where L-1 is a five-residue linker and L-2 is the longer (G4S)3 linker permitting interactions between the distal and proximal V-L/V-H domains. This 'fold-back' immunotoxin was secreted predominantly as the monomer and exhibited a 5- to 7-fold increase in bioactivity over DT390biscFv(C207) and depleted monkey T-cells in vivo.

Background. Pancreatic islet grafts are difficult to manipulate and implant in the recipient site mainly because they are formed by a group of cells suspended in a solution. This physical property determines various characteristics that are unique for pancreatic islet transplantation. The purpose of this study was to evaluate the role of fibrin glue as a delivery method for islet transplantation.Methods. C3H mouse islets were syngeneically transplanted into streptozotocin-diabetic recipients using fibrin glue in a subcutaneous pocket (Group 1) and using liquid islets injected under the kidney capsule (Group 2). Blood glucose levels were measured during 4 weeks of follow-up and compared against normal (Group 3) and diabetic levels (Group 4).Results. No statistical differences were observed between the normal, kidney capsule, and fibrin glue groups. Only the diabetic group had a statistical difference when compared with the normal control group (P <.01). At the beginning, levels in Group I (fibrin glue) were higher than in Group 2 (kidney capsule), but turned into similar values after time and no statistical differences were observed between them during follow-up.Conclusions. Islet/fibrin glue grafts placed in a subcutaneous pocket obtained the same results as liquid grafts placed under the kidney capsule, proving to be an adequate delivery method for islet transplantation and solving some of the engraftment problems we find with liquid grafts.