Islet Xenograft Survival Research Articles

Transient B-Cell Depletion Combined With Apoptotic Donor Splenocytes Induces Xeno-Specific T- and B-Cell Tolerance to Islet Xenografts

Peritransplant infusion of apoptotic donor splenocytes cross-linked with ethylene carbodiimide (ECDI-SPs) has been demonstrated to effectively induce allogeneic donor-specific tolerance. The objective of the current study is to determine the effectiveness and additional requirements for tolerance induction for xenogeneic islet transplantation using donor ECDI-SPs. In a rat-to-mouse xenogeneic islet transplant model, we show that rat ECDI-SPs alone significantly prolonged islet xenograft survival but failed to induce tolerance. In contrast to allogeneic donor ECDI-SPs, xenogeneic donor ECDI-SPs induced production of xenodonor-specific antibodies partially responsible for the eventual islet xenograft rejection. Consequently, depletion of B cells prior to infusions of rat ECDI-SPs effectively prevented such antibody production and led to the indefinite survival of rat islet xenografts. In addition to controlling antibody responses, transient B-cell depletion combined with ECDI-SPs synergistically suppressed xenodonor-specific T-cell priming as well as memory T-cell generation. Reciprocally, after initial depletion, the recovered B cells in long-term tolerized mice exhibited xenodonor-specific hyporesponsiveness. We conclude that transient B-cell depletion combined with donor ECDI-SPs is a robust strategy for induction of xenodonor-specific T- and B-cell tolerance. This combinatorial therapy may be a promising strategy for tolerance induction for clinical xenogeneic islet transplantation.

Pancreatic islet xenograft survival in mice is extended by a combination of alpha-1-antitrypsin and single-dose anti-CD4/CD8 therapy.

Clinical pancreatic islet transplantation is under evaluation for the treatment of autoimmune diabetes, yet several limitations preclude widespread use. For example, there is a critical shortage of human pancreas donors. Xenotransplantation may solve this problem, yet it evokes a rigorous immune response which can lead to graft rejection. Alpha-1-antitrypsin (AAT), a clinically available and safe circulating anti-inflammatory and tissue protective glycoprotein, facilitates islet alloimmune-tolerance and protects from inflammation in several models. Here, we examine whether human AAT (hAAT), alone or in combination with clinically relevant approaches, achieves long-term islet xenograft survival. Rat-to-mouse islet transplantation was examined in the following groups: untreated (n = 6), hAAT (n = 6, 60–240 mg/kg every 3 days from day −10), low-dose co-stimulation blockade (anti-CD154/LFA-1) and single-dose anti-CD4/CD8 (n = 5–7), either as mono- or combination therapies. Islet grafting was accompanied by blood glucose follow-up. In addition, skin xenografting was performed in order to depict responses that occur in draining lymph nodes. According to our results hAAT monotherapy and hAAT/anti-CD154/LFA-1 combined therapy, did not delay rejection day (11–24 days untreated vs. 10–22 day treated). However, host and donor intragraft inflammatory gene expression was diminished by hAAT therapy in both setups. Single dose T-cell depletion using anti-CD4/CD8 depleting antibodies, which provided 14–15 days of reduced circulating T-cells, significantly delayed rejection day (28–52 days) but did not achieve graft acceptance. In contrast, in combination with hAAT, the group displayed significantly extended rejection days and a high rate of graft acceptance (59, 61, >90, >90, >90). In examination of graft explants, marginal mononuclear-cell infiltration containing regulatory T-cells predominated surviving xenografts. We suggest that temporal T-cell depletion, as in the clinically practiced anti-thymocyte-globulin therapy, combined with hAAT, may promote islet xenograft acceptance. Further studies are required to elucidate the mechanism behind the observed synergy, as well as the applicability of the approach for pig-to-human islet xenotransplantation.

The immunobiology of pig‐to‐nonhuman primate islet xenotransplantation: insights, innovation, and impact

Previous studies of pig‐to‐non‐human primate (NHP) islet xenotransplantation have provided important insights into the immune recognition and effector pathways operative in this relevant preclinical model. The specifics of the xenograft product, microenvironment at the implantation site, and the immunosuppressive regimen significantly influence the mechanisms underlying the rejection of xenogeneic islets.Our current understanding of the immunological barriers to survival of pig islets in NHPs is largely based on studies on intraportal islet xenografts and on comparisons with islet allografts. The demonstration of cell‐mediated rejection of intraportal porcine islet xenografts at about 1 month posttransplant in monkeys immunosuppressed with the same protocols that prevent monkey islet allograft rejection indicates that islet xenograft rejection involves cellular mechanisms that are not present in acute islet allograft rejection. While these mechanisms remain poorly defined the demonstration of long‐term diabetes reversal after intraportal islet xenotransplantation in non‐human primates immunosuppressed with anti‐CD40L but not with anti‐CD40 antibody‐based protocols suggests that the therapeutic efficacy of anti‐CD40L in this transplantation setting likely involves the depletion of donor‐reactive, activated T cells besides CD40:CD40L costimulation blockade.Rejection of intraportal islet xenografts in NHPs immunosuppressed with CTLA4‐Ig and rapamycin was mediated largely by IL‐15‐primed, CXCR3+CD8+ memory T cells recruited by IP‐10 (CXCL10) positive pig islets and macrophages that showed staining for IL‐12 and iNOS. Adding basiliximab induction and tacrolimus maintenance therapy to this protocol prevented rejection in 24 of 26 recipients followed for up to 275 days. Comparison of both groups suggests, though by no means conclusive, that prolongation of graft survival in this large cohort was associated with reduced direct T cell responses to xenoantigens, reduced proportion of intrahepatic (intragraft) B cells and IFN‐γ+ and IL‐17+ CD4 and CD8 T cells, and increased local production of immunoregulatory molecules linked with Tregs, including TGF‐β, Foxp3, HO‐1, and IL‐10. Anti‐pig non‐Gal IgG antibody elicitation was suppressed in both groups.We are currently exploring the concept of negative vaccination to markedly minimize the need for immunosuppression in islet xenotransplantation. Peritransplant administration of donor apoptotic cells extended pig‐to‐mouse islet xenograft survival to >250 days when combined with peritransplant B cell‐depletion and rapamycin. This costimulation blockade‐sparing, antigen‐specific immunotherapy is expected to cause rapid pretransplant clonal deletion of indirect and anergy of direct xenospecific T cells while inducing regulatory T cells. As anti‐CD40L antibodies, B cell depleting antibodies are expected to interfere with indirect antigen presentation, costimulation, and cytokine production required for optimal T cell proliferation, memory formation, and intragraft CD8+ effector function. It is conceivable that additional strategies must be employed in NHPs and eventually in diabetic patients to achieve – as previously with anti‐CD40L antibodies – more complete, yet selective depletion of donor‐reactive, activated T‐cells for the purpose of stable xenograft acceptance.

The usefulness and limitations of the diabetic macaque model in evaluating long‐term porcine islet xenograft survival

Various groups have reported prolonged diabetes reversal and graft function after porcine islet transplantation into diabetic macaques using different experimental designs (macaque source, islet source, type of immunosuppression): subsequently, the International Xenotransplantation Association has published recommendations for entering a clinical trial. Our experiments showed limitations that affected consistent achievement of long-term survival. We aimed to identify these limitations and underlying causes to emphasize the translational value of this highly relevant type 1 diabetic macaque model. We reviewed data from our institution and literature data on long-term porcine islet xenograft survival in the diabetic macaque model, especially focusing on aspects of incomplete diabetes reversal relative to macaque normal values. This phenomenon was compared with diabetes reversal in an allo-islet transplant model in macaques and with chronic insulin treatment of diabetic macaques, all with 180-day follow-up. This comparison enabled to identify potential model limitations and underlying causative factors. Especially in the xenograft model, the achievement of long-term graft survival revealed limitations including chronic, mild hyperglycemia and absence of body weight (BW) gain or even progressive BW loss. Metabolic incompatibilities in glycemic control (i.e., insulin kinetics) between the pig and macaque species underlie chronic, mild hyperglycemia. This phenomenon might not bear relevance for the pig-to-human species combination because the glycemic control in pigs and humans is similar and differs from that in nonhuman primates (NHP). Weight loss could be related to changes in the gastrointestinal tract related with local high exposure to orally administered immunosuppressants; these must be given at higher dose levels because of low bioavailability in macaques to achieve systemic exposure at therapeutic levels. This is aggravated by insufficient graft insulin production in proportion to the needs of macaques: this model limitation has no translational value to the pig-to-human setting. Nutritional deficits can result in incorrect interpretation of blood glucose levels and C-peptide levels regarding graft function. Likewise, nutritional status alters physiologic responses, influencing susceptibility to infectious and noninfectious complications. THE model-induced confounding described interferes with accurate interpretation of safety and efficacy studies, which affects the translational value of pig-to-NHP islet cell transplant studies to the pig-to-human transplant condition.

The Effect of Co-Stimulation Blockade on Memory T Cell Responses in a Rat-To-Mouse Islet Xenotransplantation Model

Background: Co-stimulatory signals have been reported to play an important role in islet xenograft rejection. Co-stimulation blockade cannot inhibit graft rejection in sensitized recipients. We investigated whether pre-existing donor-specific antibodies influenced islet xenograft survival in recipients treated with co-stimulation blockade. Methods: MaleC57BL/6 mice with alloxan-induced diabetes were used as recipients and male Lewis rats as donors. Xenoimmune serum was collected from mice transplanted with 200 Lewis rat islets and used as source of immune antibodies. Diabetic naïve C57BL/6 mice were transplanted under the left kidney capsule with 100 Lewis rat islets and the recipients were divided into three groups receiving: i) isotype control antibodies; ii) anti-CD154 and CTLA4Ig; and iii) anti-CD 154, CTLA4Ig, and anti-LFA-1 every second day from day 0 to day 8. Half of the animals in each group received naïve mouse serum (as source of control antibody) and half xenoimmune serum at the time of transplantation. Graft survival was followed by daily recordings of non-fasting blood glucose levels and body weight. Cured mice were examined by the IPGT test at 4 and 6-8 weeks after transplantation. Results: Anti-donor antibody was detected in the immune antibody injected recipient serum at 48 hours after transplantation but disappeared after 96 hour. There was no significant difference in graft survival between immune antibody and naive antibody injected groups. Mice receiving isotype control antibodies rejected their grafts within 13 days, and histology showed severe infiltration of both PMN and lymphocytes with few or no remaining grafts. The graft function in mice receiving double and triple treatment survived longer (>70 days), however, approximately half of them lost graft function after 100 days. In cured mice, islet grafts were verified by histological examination. Most of them showed mononuclear cell infiltration around the grafts. In mice rejecting the grafts, no or very little graft mass was seen with severe infiltration of mononuclear cells and some PMNs. In the IPGTT at 6-8 weeks after transplantation, return of normoglycemia was significantly delayed compared to the response in the IPGTT done at 4 weeks, indicating a gradual graft loss during the observation period. Mixed lymphocyte cultures in vitro showed no significant difference in T-cell proliferation between islet rejecting and accepting hosts. Anti-donor antibodies developed even in mice maintaining graft function. Conclusion: Xenoimmune antibody alone did not affect rat islet xenograft survival in naïve mice when their T cell immunity was impaired through co-stimulation blockade. Co-stimulation blockade could not induce permanent graft survival in the rat-to-mouse islet xenotransplantation model.

Adenovirus-mediated heme oxygenase-1 gene transfer to neonatal porcine islet-like cluster cells: the effects on gene expression and protection from cell stress

Porcine islet xenotransplantation is a promising strategy for the treatment of diabetes that overcomes donor shortages. However, islet xenografts are susceptible to oxidative stress and apoptosis. Heme oxygenase-1 (HO-1) has been shown to protect cells from oxidative stress, apoptosis and inflammation. Here, we investigated whether introduction of human HO-1 (hHO-1) into neonatal porcine islet-like cell clusters (NPCCs) can induce beneficial transcriptional changes in NPCCs against cellular stress. NPCCs were transduced with either adenovirus-HO-1 (Ad-HO-1) or control adenovirus-GFP (Ad-GFP). After treatment with hydrogen peroxide (H2O2) for 24 hours, nitrite oxide (NO) production assays were performed to detect oxidative stress. Microarray analysis was performed using a pig oligonucleotide 44 K gene chip. We profiled transcriptional changes to apoptosis, oxidant and inflammatory genes, and real-time PCR analysis was also performed to confirm the microarray results. Survival of NPCCs after treatment with H2O2 was significantly higher in the Ad-HO-1 group (p<0.001), and NO production also decreased in the Ad-HO-1 group (p<0.01). The microarray results showed that the expression of pro-apoptosis genes such as CASP3, CASP7, CASP10, CIDE-B and CIDE-C was significantly decreased in the Ad-HO-1 virus group (CASP10; p<0.05, CASP3, CIDE-C; p<0.01, CASP7, CIDE-B; p<0.001). We also found that the expression of oxidative stresses genes including COX1, COX2, CYB5A, SDHD and NOS2 was decreased, and that the anti-oxidant genes Gpx1 and SOD2 were increased in the Ad-HO-1 group (NOS2; p<0.05, COXI, COX2, CYB5A, SDHD, SOD2, GPX1; p<0.001). However, inflammatory gene expression was not significantly changed. Realtime PCR analysis confirmed the results of the microarray analysis. These results shed light on the underlying mechanisms of the protective effects of hHO-1 on porcine islets from cellular stresses and suggest that hHO-1 could be a promising target gene for the production of transgenic pigs that confer improved islet xenograft survival.

Tracing of islet graft survival by way of in vivo fluorescence imaging

To increase the success rate in xenogeneic islet transplantation, proper assessment of graft mass is required following transplantation. For this reason, we aimed to develop a suitable fluorescence imaging system to monitor islet xenograft survival in diabetic mice. Adenovirus vector encoding enhanced green fluorescent protein-transduced rat pancreatic islets were transplanted under the renal capsule of streptozotocin-induced diabetic mice and the fluorescence signal was quantified over time using a cooled charge-coupled device. Non-fasting blood glucose levels were recorded during the same period. Insulin release from transduced and control islets was detected via enzyme-linked immunosorbent assay. Adenovirus vector encoding enhanced green fluorescent protein infection did not alter the function or survival of pancreatic islets post transduction. A direct correlation was found between the number of islets (250-750) transplanted under the kidney capsule and the blood glucose recovery. Fluorescence imaging appears to be a useful tool for quantitative assessment of islet cell viability post transplantation and could permit earlier detection of graft rejection.

Effects of Gene Transfer CTLA4Ig and Anti-CD40L Monoclonal Antibody on Islet Xenograft Rejection in Mice

Blockade of a costimulatory pathway by adenovirus-mediated cytotoxic T lymphocyte associated antigen 4 immunoglobulin (CTLA4-Ig) gene transfer and anti-CD40L mAb(MR1) have been reported to enhance graft survival in several experimental transplantation models. In this study, we investigated the effects of gene transfer of CTLA4Ig and MR1 on islet xenograft rejection in mice. Recombinant adenovirus AdCTLA4Ig was constructed to express CTLA4Ig. Islet grafts from adult male DA rats transferred with AdCTLA4Ig were transplanted to streptozocin-induced diabetic Balb/c mice. The diabetic mice were treated with MR1 after transplantation. We evaluated the islet xenograft mean survival time as well as changes in interleukin-2 (IL-2) and tumor necrosis factor-alpha (TNF-α) levels in transplanted mice. The mean survival of islet xenografts in the MR1 treatment group was 34.9 ± 5.62 days, in the AdCTLA4Ig treatment group it was 56.5 ± 10.64 days, and in the AdCTLA4Ig plus MR1 treatment group it was 112.9 ± 19.26 days, all significantly prolonged compared with an untreated group (8.1 ± 0.83 days). Within 1 week after transplantation the levels of IL-2 and TNF-α showed sharp increases in the untreated group, being significantly higher than those observed prior to transplantation. In conclusion, using both AdCTLA4Ig and MR1 can improve the islet xenograft survival. The beneficial effects of the combined use of the 2 reagents were superior to either 1 alone, possibly related to down-regulated expression of Th1 cell–related cytokines.

Anti-CD154 mAb and Rapamycin Induce T Regulatory Cell Mediated Tolerance in Rat-to-Mouse Islet Transplantation

BackgroundAnti-CD154 (MR1) monoclonal antibody (mAb) and rapamycin (RAPA) treatment both improve survival of rat-to-mouse islet xenograft. The present study investigated the effect of combined RAPA/MR1 treatment on rat-to-mouse islet xenograft survival and analyzed the role of CD4+CD25+Foxp3+ T regulatory cells (Treg) in the induction and maintenance of the ensuing tolerance.Methodology/Principal FindingsC57BL/6 mice were treated with MR1/RAPA and received additional monoclonal anti-IL2 mAb or anti CD25 mAb either early (0–28 d) or late (100–128 d) post-transplantation. Treg were characterised in the blood, spleen, draining lymph nodes and within the graft of tolerant and rejecting mice by flow cytometry and immunohistochemistry. Fourteen days of RAPA/MR1 combination therapy allowed indefinite islet graft survival in >80% of the mice. Additional administration of anti-IL-2 mAb or depleting anti-CD25 mAb at the time of transplantation resulted in rejection (100% and 89% respectively), whereas administration at 100 days post transplantation lead to lower rejection rates (25% and 40% respectively). Tolerant mice showed an increase of Treg within the graft and in draining lymph nodes early post transplantation, whereas 100 days post transplantation no significant increase of Treg was observed. Rejecting mice showed a transient increase of Treg in the xenograft and secondary lymphoid organs, which disappeared within 7 days after rejection.Conclusions/SignificancesThese results suggest a critical role for Treg in the induction phase of tolerance early after islet xenotransplantation. These encouraging data support the need of developing further Treg therapy for overcoming the species barrier in xenotransplantation.

Natural killer T cell facilitated engraftment of rat skin but not islet xenografts in mice

We have studied cellular components required for xenograft survival mediated by anti-CD154 monoclonal antibody (mAb) and a transfusion of donor spleen cells and found that the elimination of CD4(+) but not CD8(+) cells significantly improves graft survival. A contribution of other cellular components, such as natural killer (NK) cells and natural killer T (NKT) cells, for costimulation blockade-induced xenograft survival has not been clearly defined. We therefore tested the hypothesis that NK or NKT cells would promote rat islet and skin xenograft acceptance in mice. Lewis rat islets or skin was transplanted into wild type B6 mice or into B6 mice that were Jalpha18(null), CD1(null), or beta2 microglobulin (beta2M)(null) NK 1.1 depleted, or perforin(null). Graft recipients were pretreated with an infusion of donor derived spleen cells and a brief course of anti-CD154 mAb treatments. Additional groups received mAb or cells only. We first observed that the depletion of NK1.1 cells does not significantly interfere with graft survival in C57BL/6 (B6) mice. We used NKT cell deficient B6 mice to test the hypothesis that NKT cells are involved in islet and skin xenograft survival in our model. These mice bear a null mutation in the gene for the Jalpha18 component of the T-cell receptor. The component is uniquely associated with NKT cells. We found no difference in islet xenograft survival between Jalpha18(null) and wild type B6 mice. In contrast, median skin graft survival appeared shorter in Jalpha18(null) recipients. These data imply a role for Jalpha18(+) NKT cells in skin xenograft survival in treated mice. In order to confirm this inference, we tested skin xenograft survival in B6 CD1(null) mice because NKT cells are CD1 restricted. Results of these trials demonstrate that the absence of CD1(+) cells adversely affects rat skin graft survival. An additional assay in beta2M(null) mice demonstrated a requirement for major histocompatibility complex (MHC) class I expression in the graft host, and we demonstrate that CD1 is the requisite MHC component. We further demonstrated that, unlike reports for allograft survival, skin xenograft survival does not require perforin-secreting NK cells. We conclude that MHC class I(+) CD1(+) Jalpha18(+) NKT cells promote the survival of rat skin but not rat islet xenografts. These studies implicate different mechanisms for inducing and maintaining islet vs. skin xenograft survival in mice treated with donor antigen and anti-CD154 mAb, and further indicate a role for NKT cells but not NK cells in skin xenograft survival.

In Vivo Controlling of Cellular Response to Pig Islet Xenografts by Adenovirus-Mediated Expression of Either Membrane-Bound Human FasL or Human Decoy Fas

The critical problem with clinical islet transplantation for patients with type 1 diabetes is the severe shortage of human donors. Pig islet xenotransplantation has the potential to provide a virtually unlimited source of donor pancreata. However, our previous studies demonstrated that cell-mediated rejection, especially human CD8 + cytotoxic T lymphocyte (CTL)–mediated cytotoxicity, remains a major obstacle for long-term islet xenograft survival. Moreover, we have demonstrated that the overexpression of either membrane-bound human FasL (mFasL) or human decoy Fas antigen (decoy Fas) in pig islets not only prevented CTL xenocytotoxicity in vitro, but also prolonged histological survival of pig islet xenografts in vivo. Therefore, the aim of the present study was to determine whether adenoviral transfer of these genes into pig islets ex vivo prior to transplantation had a beneficial effect on posttransplantation glycemic control of diabetic recipients. Isolated pig islets were transfected with adenovirus vector carrying complementary DNA (cDNA) of either mFasL or decoy Fas. The transfected islets were transplanted under the kidney capsule of diabetic recipient rats. Rats transplanted with either mFasL- or decoy Fas-transfected pig islet grafts showed significantly suppressed blood glucose levels from 12 hours to 18 hours posttransplantation compared with control groups transplanted with empty vector-transfected pig islets. Unfortunately, blood glucose levels of these groups were increased, with no significant difference observed at 24 hours posttransplantation. However, transgenic expression of these molecules with clinically tolerable amount of immunosuppressants may be more effective to achieve islet xenograft survival in the future.

Adenovirus-Mediated Gene Expression of the Human c-FLIPL Gene Protects Pig Islets Against Human CD8 + Cytotoxic T Lymphocyte-Mediated Cytotoxicity

Cell-mediated immunity, especially of human CD8+ cytotoxic T lymphocytes (CTLs) is believed to have an important role in the long-term survival of pig islet xenografts. Protection against human CD8+ CTL cytotoxicity may reduce the direct damage to pig islets and enable long-term xenograft survival in pig-to-human islet xenotransplantation. We have previously reported that c-FLIP S/L genes, which are potent inhibitors of death receptor–mediated proapoptotic signals through binding competition with caspase-8 for recruitment to the Fas-associated via death domain (FADD), markedly suppress human CD8+ CTL-mediated xenocytotoxicity. In addition, the cytoprotective effects of c-FLIP L seem to be significantly stronger than those of c-FLIP S . Accordingly, in the present study, expression of c-FLIP L was induced in intact pig islets by adenoviral transduction. Consequently, the cytoprotective capacity of the transgene in pig islets was examined in in vitro and in vivo exposure to human CD8+ CTLs. Cells from untransduced islets or mock islets were sensitive to CD8+ CTL-mediated lysis (59.3% ± 15.9% and 64.0% ± 8.9% cytotoxicity, respectively). In contrast, cells from pig islets transduced with the c-FLIP L gene were markedly protected from lysis (30.5% ± 3.5%). Furthermore, prolonged xenograft survival was elicited from pig islets transduced with this molecule as assessed using an islet transplant model using the rat kidney capsule. Thus, these data indicate that intact pig islets can be transduced to express c-FLIP L with adenovirus. Pig islets expressing c-FLIP L are significantly resistant to human CTL killing and further exhibit beneficial effects to prolong xenograft survival.

Monotherapy with Anti-LFA-1 Monoclonal Antibody Promotes Long-Term Survival of Rat Islet Xenografts

Previously we demonstrated that anti-LFA-1 monoclonal (mAb) could promote long-term survival of discordant porcine islet xenografts in mice. The aim of this study, therefore, was to determine whether a shortterm administration of anti-LFA-1 mAb would promote long-term survival of concordant rat islet xenografts in mice, and whether combining short-term administration of anti-LFA-1 mAb therapy with an immunosuppressive drug, rapamycin, would facilitate islet xenograft survival. Streptozotocin-induced diabetic BALB/c mice were transplanted with 500 Wistar-Furth rat islets under the kidney capsule and were either left untreated or treated with short-term administration of rapamycin (0.2 mg/kg) alone, anti-LFA-1 mAb (0.2 mg/ dose) alone, or a combination of rapamycin and anti-LFA-1 mAb using the same doses. All untreated mice rejected their grafts by 24 days posttransplantation with a mean graft survival time of 18.8 +/- 2.5 days posttransplantation (n = 5). All mice treated with rapamycin alone had prolonged islet graft survival but eventually rejected their islet grafts by 81 days posttransplantation. In contrast, the majority of the mice (27/ 28) treated with anti-LFA-1 mAb alone maintained long-term normoglycemia (>100 days). Rapamycin in combination with anti-LFA-1 mAb proved equally effective with 29 of 30 mice maintaining normoglycemia for more than 100 days posttransplantation. Low levels of mouse anti-rat antibodies, as well as a decrease in the degree of mononuclear cell infiltration of the islet graft, closely correlated with long-term islet xenograft survival. These results demonstrate that monotherapy with anti-LFA-1 mAb is highly effective in promoting long-term survival of rat islet xenografts and that combination of anti-LFA-1 mAb with rapamycin does not facilitate nor abrogate the induction of long-term xenograft survival by anti-LFA-1 mAb therapy in BALB/c mice. Our study indicates that immunomodulation through mAb therapy could form a significant component of future antirejection therapies in clinical islet xenotransplantation.

Mitomycin-C Treatment Followed by Culture Produces Long-Term Survival of Islet Xenografts in a Rat-to Mouse Model

One of the goals of islet transplantation is to transplant viable islets without host immunosuppression. The present study was designed to determine whether pretreatment of islets with mitomycin-C (MMC) followed by culture enhances islet survival in a rat-to-mouse xenogeneic combination. WS(RT1k) rat islets pretreated with various concentrations of MMC (0, 3.2, 10, 32, 100, 320, and 1000 microg/ml) were tested for viability by in vitro insulin secretory capacity and vital staining of islets. The MMC-treated islets (10 microg/ml) cultured for various periods (4, 20, or 40 h, 3 or 7 days) were transplanted into the renal subcapsular space of STZ-induced diabetic C57BL/6 (B6: H-2b) mice. MMC-treated or nontreated islets were subjected to microarray gene analysis and immunohistological study. Evaluation of in vitro insulin secretory capacity and vital staining of islets indicated that MMC at a dose < or =32 microg/ml is nontoxic and preserves islet function. Marked prolongation of graft survival was noted with half of islet grafts surviving indefinitely (>100 days) when 10 microg/ml of MMC-treated islets was transplanted after 40 h or 3 days in culture, but not when they were transplanted within 4 h following treatment or at 7 days following treatment, indicating that there is a critical culture period necessary for successful islet graft survival. Microarray analysis suggested possible genes for this prolongation with TGF-beta highly expressed in MMC-treated islets subjected to culture for 3 days. Our results indicate that MMC treatment followed by a critical culture period induces marked prolongation of rat islet xenograft survival in nonimmunosuppressed recipient mice, offering a strategy for islet transplantation without immunosuppression.

Islet xenotransplantation: current status of preclinical studies in the pig-to-nonhuman primate model

Porcine islets are a major focus of current research in nonhuman primate xenotransplantation models. Major advances have been obtained recently and these are briefly described. Reports by three independent centres have described 6-month porcine islet xenograft survival in nonhuman primates. Two of these have obtained such results by interfering with the CD40/CD154 co-stimulatory pathway. While these results are groundbreaking, the immunosuppressive regimens used are not viewed as clinically applicable and will need to be modified before islet xenotransplantation can be considered for clinical trials. In contrast, preliminary results by Gianello and colleagues have demonstrated 6-month survival of diabetic nonhuman primates transplanted subcutaneously with encapsulated porcine islets, in the complete absence of immunosuppression. The confirmation and full assessment of these results are eagerly awaited. Importantly, to date, no evidence of xenozoonoses has been observed following porcine islet xenotransplantation to nonhuman primates. Of the possible organ candidates for xenotransplantation, porcine islets are the closest to a possible clinical application. Prior to the initiation of ethical and safe clinical trials, however, further efforts to generate additional efficacy and safety data in the nonhuman primate model will be indispensable.

Long-Term Survival and Function of Intraportal Porcine and Human Islet Xenografts in Nondiabetic Nude Mice

Instant blood-mediated inflammatory reaction (IBMIR) is a serious obstacle to both clinical islet allotransplantation and future islet xenotransplantation via the portal vein. We have previously observed uniform long-term tilapia (fish) islet xenograft survival when islets were transplanted intraportally into nondiabetic nude mice (nDNM), but not in diabetic nude mice (DNM). In this study, we examined whether human islets (HI) and adult porcine islets (API) can tolerate intraportal transplantation into nDNM like tilapia islets. HI and API were transplanted intraportally into nDNM. Recipients were humanely killed either 14 or 28 days after transplantation and livers were processed for histology. Human insulin and human C-peptide were measured in the terminal serum samples of HI recipients. In six of seven HI and seven of seven API recipients, liver histology showed insulin-positive islet xenografts. In recipients with HI, the numbers of islets/ductal structures seen histologically correlated well with serum sample results. These results show that HI and API can survive and function long term after intraportal transplantation into nDNM recipients. Our previous and present data indicated that DNM and nDNM could be useful models to study “glucose toxicity” and the role of IBMIR in the fate of intraportal islet grafts.

Both Innate and Adaptive Major Histocompatibility Complex Class I-Dependent Immunity Impair Long-Term Islet Xenograft Survival

Natural killer (NK) cells have long been appreciated for their rapid, proinflammatory contribution to host defense. However, more recent studies show an unexpected regulatory role for host major histocompatibility complex (MHC) class I-dependent immunity and NK cells in promoting tolerance induction to islet allografts. It is unclear whether the potential tolerance induction to islet xenografts follows similar requirements to those found in allograft tolerance. In this study, we determined whether induced islet xenograft prolongation also showed a reliance on MHC class I-dependent immune pathways. In particular, we tested whether NK1.1 + cells and/or CD8 T cells were required for the long-term islet xenograft survival in a rat-to-mouse transplant model. Short-term host treatment with combined anti-CD154 plus anti-LFA-1 (CD11a) resulted in prolonged, but not indefinite, survival of WF rat islet xenografts in C57BI/6 mouse recipients. In stark contrast with similar islet allograft studies, adjunct treatment with anti-NK1.1 therapy combined wither anti-CD154/anti-LFA-1 treatment led to long-term (>100 days) survival of the majority of islet xenografts. In parallel studies, we determined whether CD8 T cells also contributed a barrier to xenograft survival. Similar to results found in anti-NK1.1–treated animals, CD8-deficient (knockout) recipients also demonstrated augmented xenograft prolongation after combined anti-CD154/anti-LFA-1 therapy. Taken together, NK1.1 + cells (NK/NKT cells) and CD8 T cells constitute differing MHC class I-dependent immune pathways forming a significant barrier to xenograft prolongation.

Anti‐CD40LAb combined with CTLA4Ig prolonged porcine islet xenograft survival indefinitely in GalT(+/+) C57BL/6 mice but not indefinitely in syngeneic GalT(−/−) mice

Defining the immune response to xenogeneic pancreatic islet graft and induction of immune tolerance are crucial for the successful islet xenotransplantation. Galactosyltransferase‐deficient(GalT‐KO) mice provide a unique opportunity to study xenograft rejection in the context of anti‐α1,3‐Gal antibody(α‐Gal Ab) response. We investigated the rejection mechanism of porcine islet xenograft in C57BL/6 GalT‐KO mice. 5000 islet equivalent of pig islets isolated from CMS miniature pig were transplanted under the kidney capsule of diabetic GalT‐KO or normal B6 mice. Graft rejection occurred earlier in GalT‐KO(mean survival day(MSD)=7) than in normal mice(MSD=15). Heavy infiltration of CD4+ and CD8+T cells in rejected graft implicated T cells as major player in graft rejection. Notably, intra‐graft infiltration of polymorphonuclear leukocytes with CD4+ T cells in peri‐islet on DPT 3 was observed only in GalT‐KO recipients, which was abolished by CD4+T cell depletion. α‐CD40LmAb+CTLA4Ig prolonged graft survival indefinitely in wild type(&gt;200 days), but not indefinitely in GalT‐KO mice(MSD=60) where the rising titer of α‐Gal Ab was observed. Conclusively, the control of α‐Gal Ab and its subsequent immune response in addition to T cell would be required for immunological tolerance in discordant islet xenotransplantation such as pig to GalT‐KO mice or pig to human islet xenotransplantation.

Lack of amyloidogenicity and altered prohormone processing of porcine islet amyloid polypeptide may promote function and survival of pig islet xenografts.

Lack of amyloidogenicity and altered prohormone processing of porcine islet amyloid polypeptide may promote function and survival of pig islet xenografts.

Anti-CD25 mAb, Anti-IL2 mAb, and IL2 Block Tolerance Induction Through Anti-CD154 mAb and Rapamycin in Xenogeneic Islet Transplantation

Background We have used anti-CD154 monoclonal antibody (mAb; MR1) and rapamycin (rapa) to induce tolerance to islet xenografts. The aim of this study was to investigate whether classical anergy and/or regulation by interleukin (IL)2-dependent CD25 + T regulatory cells played roles in the induction and maintenance of tolerance in this model. Methods Streptozotocin-induced diabetic mice were transplanted with rat islets. We performed the following groups: control group, islet transplantation without therapy; rapamycin group, 0.2 mg/kg by oral gavage on days 0, 1, 2, and every other day to day 14; anti-CD154 mAb (MR1) group, 0.5 mg intraperitoneally on days 0, 2, and 4; combination therapy group with rapa and MR1. We then administered in addition to the combination therapy with early (from days 0 to 14 [for IL2] or to 28 [for anti-IL2 mAb and anti-CD25 mAb] post-transplantation) or late (from days 100 to 114 [for IL2] or to 128 [for anti-IL2 mAb and anti-CD25 mAb] posttransplantation) recombinant IL2 (2000 U, intraperitoneally twice a day), a neutralizing anti-IL2 mAb (S4B6-1, 0.3 mg intraperitoneally twice weekly), and a depleting anti-CD25 mAb (PC61, 0.3 mg intraperitoneally twice weekly), respectively. Histology was performed at time of rejection. Results Rapa and MR1 therapy alone significantly prolonged xenograft survival compared to the control group: median graft survival was 34 days versus 17 days ( P < .05) and 98 days versus 17 days ( P < .05), respectively, but rejection still occurred. Combination therapy with MR1 and rapa allowed indefinite graft survival (median graft survival [MGS] >200 days, P < .001). When exogenous IL2 was administered early with MR1 and rapa, rapid rejection developed in 18 of 18 mice (MGS 7 days), whereas when IL2 was given late, only 3 of 10 developed rejection. Early administration of anti-IL2 mAb led to rejection in 10 of 10 mice (MGS 42 days), whereas late administration led to rejection in only one of four mice. Early administration of anti-CD25 mAb led to rejection in eight of nine mice (MGS 49 days), whereas late administration led to rejection in only three of seven mice. Conclusions Rapa and MR1 allowed indefinite graft survival of islet xenografts. Classical anergy and regulation by IL2-dependent CD25 + T regulatory cells were critical in the induction of tolerance in the immediate posttransplantation period and less important for maintenance of tolerance.