Marilyne Coulombe

CD4-Dependent Generation of Dominant Transplantation Tolerance Induced by Simultaneous Perturbation of CD154 and LFA-1 Pathways

CD154 and LFA-1 (CD11a) represent conceptually distinct pathways of receptor/ligand interactions (costimulation and adhesion/homing, respectively) that have been effectively targeted to induce long-term allograft acceptance and tolerance. In the current study, we determined the relative efficacy and nature of tolerance induced by mAbs specific for these pathways. In vitro analysis indicated that simultaneous targeting of CD154 and LFA-1 resulted in profound inhibition of alloreactivity, suggesting that combined anti-CD154/anti-LFA-1 therapy could be highly effective in vivo. Thus, we evaluated combining mAb therapies targeting CD154 and LFA-1 for inducing transplantation tolerance to pancreatic islet allografts. Monotherapy with either anti-CD154 or anti-LFA-1 was partially effective for inducing long-term allograft survival, whereas the combination resulted in uniform allograft acceptance in high-responder C57BL/6 recipients. This combined therapy was not lymphocyte depleting and did not require the long-term deletion of donor-reactive T lymphocytes to maintain allograft survival. Importantly, combined anti-CD154/anti-LFA therapy uniquely resulted in “dominant” transplantation tolerance. Therefore, simultaneous perturbation of CD154 and LFA-1 molecules can result in profound tolerance induction not accomplished through individual monotherapy approaches. Furthermore, results show that such regulatory tolerance can coexist with the presence of robust anti-donor reactivity, suggesting that active tolerance does not require a corresponding deletion of donor-reactive T cells. Interestingly, although the induction of this regulatory state was highly CD4 dependent, the adoptive transfer of tolerance was less CD4 dependent in vivo.

Donor antigen-presenting cell-independent rejection of islet xenografts

Donor-derived antigen-presenting cells (APC) are thought to serve as major stimulators for triggering the rejection of tissue allografts. However, the capacity of APC to stimulate xenogeneic T cells is generally deficient relative to the corresponding response from allogeneic T cells. For this reason, the contribution of donor-type APC to xenogeneic graft rejection remains unclear. Using a concordant species combination (rat to mouse), we examined the requirement for donor-type APC in triggering islet xenograft rejection. While the depletion of donor-type APC resulted in indefinite allograft survival, similar depletion of APC from xenogeneic rat islets resulted in only modest graft prolongation. Furthermore, APC-depleted rat xenografts were rejected by a CD8+ T cell-independent mechanism, as determined by appropriate depletion of T cell subsets through monoclonal antibody therapy. This contrasts with the dependence of islet allograft rejection on both CD4+ and CD8+ T cells. Although in vitro experiments show that rat APC can directly stimulate mouse T cells, rat APC do not appear to be required for xenograft immunity in vivo. We conclude that the mechanisms of islet allograft and xenograft rejection differ both in the dependence on donor-type APC and in the role of T cell subsets in the response.

Redox Modulation Protects Islets From Transplant-Related Injury

OBJECTIVE Because of reduced antioxidant defenses, β-cells are especially vulnerable to free radical and inflammatory damage. Commonly used antirejection drugs are excellent at inhibiting the adaptive immune response; however, most are harmful to islets and do not protect well from reactive oxygen species and inflammation resulting from islet isolation and ischemia-reperfusion injury. The aim of this study was to determine whether redox modulation, using the catalytic antioxidant (CA), FBC-007, can improve in vivo islet function post-transplant. RESEARCH DESIGN AND METHODS The abilities of redox modulation to preserve islet function were analyzed using three models of ischemia-reperfusion injury: 1) streptozotocin (STZ) treatment of human islets, 2) STZ-induced murine model of diabetes, and 3) models of syngeneic, allogeneic, and xenogeneic transplantation. RESULTS Incubating human islets with catalytic antioxidant during STZ treatment protects from STZ-induced islet damage, and systemic delivery of catalytic antioxidant ablates STZ-induced diabetes in mice. Islets treated with catalytic antioxidant before syngeneic, suboptimal syngeneic, or xenogeneic transplant exhibited superior function compared with untreated controls. Diabetic murine recipients of catalytic antioxidant–treated allogeneic islets exhibited improved glycemic control post-transplant and demonstrated a delay in allograft rejection. Treating recipients systemically with catalytic antioxidant further extended the delay in allograft rejection. CONCLUSIONS Pretreating donor islets with catalytic antioxidant protects from antigen-independent ischemia-reperfusion injury in multiple transplant settings. Treating systemically with catalytic antioxidant protects islets from antigen-independent ischemia-reperfusion injury and hinders the antigen-dependent alloimmune response. These results suggest that the addition of a redox modulation strategy would be a beneficial clinical approach for islet preservation in syngeneic, allogeneic, and xenogeneic transplantation.

Pancreatic Islet Allograft Immunity and Tolerance: the Two‐Signal Hypothesis Revisited

The principle assumption of this discussion is that costimulation (CoS) forms the primary stimulus that compels T cells to mount a response to their specific antigen. However, this response can be either positive or negative, depending on the developmental stage of the T cell and the microenvironment in which the antigen and CoS are received. Thus, both immunity and tolerance may represent different outcomes of a two-signal process. We would emphasize that CoS is a functional term and not a strict molecular definition. While many molecular interactions have been described as providing CoS activity, notably those involving the B-7 family of cell surface molecules, it is not yet clear what combination(s) of non-antigen-specific signals may fulfil this function. This point is important because many studies have achieved tolerance through strategies designed to inhibit specific CoS molecules. However, it may be that differential signaling through distinct CoS molecules, rather than a global inhibition of CoS per se, plays a role in the generation of active tolerance in such studies (Bluestone 1995). A corollary of this notion is that antigen (signal 1) delivery to T cells is a null event and so is not an inherently paralysing signal. Of course, if signal 1 is not itself a tolerogenic signal, then other mechanisms are necessary to explain many empirical observations of tolerance to allogeneic or self antigens. This is best illustrated by those forms of functional tolerance to either alloantigens or self antigens that do not appear to be the result of clonal deletion/inactivation. It would be relatively simple to invoke a model of tolerance whereby the relevant tissue-destructive cell is eliminated or inactivated; such a model would preclude the necessity to suggest active regulatory mechanisms of tolerance. However, in several model systems, including our own observations concerning tolerance induction to APC-depleted islet allografts, tissue-destructive T cells can persist in recipients tolerant to allogeneic or self antigens. Furthermore, there are key examples in which tolerance demonstrates a dominant phenotype; that is, tolerant cells can regulate the activity of naive, non-tolerant cells. This latter observation points to the function of an active, regulatory form of tolerance. As such, we would emphasize that tolerance should not be defined as unresponsiveness since the tolerant state is the consequence of very active immune reactions.

Prolongation of Islet Xenograft Survival by Cryopreservation

Our attempt to reduce islet immunogenicity by slow cooling to −40°C, storage at −196°C, and rapid thawing is based on the differential susceptibility of various cell types to a freeze-thaw process. Five hundred rat islets (≥10 μm) were immediately implanted or cryopreserved and then implanted beneath the renal capsule of streptozocin-induced diabetic mice with or without an injection of anti-lymphocyte serum at the time of transplantation. Thirteen days after transplantation, all fresh xenografts had rejected, whereas 37.5% of cryopreserved grafts were still functioning. In immunosuppressed mice, 6.2% of fresh xenografts and 54.5% of cryopreserved grafts were functioning 19 days after transplantation. These results show that cryopreservation can extend xenograft survival.

Mannan-Binding Lectin–Associated Serine Protease 1/3 Cleavage of Pro–Factor D into Factor D In Vivo and Attenuation of Collagen Antibody-Induced Arthritis through Their Targeted Inhibition by RNA Interference–Mediated Gene Silencing

The complement system is proposed to play an important role in the pathogenesis of rheumatoid arthritis (RA). The complement system mannan-binding lectin–associated serine proteases (MASP)-1/3 cleave pro–factor D (proDf; inactive) into Df (active), but it is unknown where this cleavage occurs and whether inhibition of MASP-1/3 is a relevant therapeutic strategy for RA. In the present study, we show that the cleavage of proDf into Df by MASP-1/3 can occur in the circulation and that inhibition of MASP-1/3 by gene silencing is sufficient to ameliorate collagen Ab–induced arthritis in mice. Specifically, to examine the cleavage of proDf into Df, MASP-1/3–producing Df−/− liver tissue (donor) was transplanted under the kidney capsule of MASP-1/3−/− (recipient) mice. Five weeks after the liver transplantation, cleaved Df was present in the circulation of MASP-1/3−/− mice. To determine the individual effects of MASP-1/3 and Df gene silencing on collagen Ab–induced arthritis, mice were injected with scrambled, MASP-1/3–targeted, or Df-targeted small interfering RNAs (siRNAs). The mRNA levels for MASP-1 and -3 decreased in the liver to 62 and 58%, respectively, in mice injected with MASP-1/3 siRNAs, and Df mRNA decreased to 53% in the adipose tissue of mice injected with Df siRNAs; additionally, circulating MASP-1/3 and Df protein levels were decreased. In mice injected with both siRNAs the clinical disease activity, histopathologic injury scores, C3 deposition, and synovial macrophage/neutrophil infiltration were significantly decreased. Thus, MASP-1/3 represent a new therapeutic target for the treatment of RA, likely through both direct effects on the lectin pathway and indirectly through the alternative pathway.

Differential Impact of Chronic Hyperglycemia on Humoral Versus Cellular Primary Alloimmunity

Diabetes is prevalent among solid organ transplant recipients and is universal among islet transplant recipients. Whereas diabetes is often considered to result in an immune-compromised state, the impact of chronic hyperglycemia on host alloimmunity is not clear. Potential immune-modifying effects of obesity, autoimmunity, or diabetogenic agents like streptozotocin may confound understanding alloimmunity in experimental models of diabetes. Therefore, we sought to determine the role of chronic hyperglycemia due to insulinopenia on alloimmunity using the nonautoimmune, spontaneously diabetic H-2b–expressing C57BL/6 Ins2Akita mice (Akita). Akita mice harbor a mutated Ins2 allele that dominantly suppresses insulin secretion, resulting in lifelong diabetes. We used BALB/c donors (H-2d) to assess alloimmunization and islet transplantation outcomes in Akita recipients. Surprisingly, chronic hyperglycemia had little effect on primary T-cell reactivity after alloimmunization. Moreover, Akita mice readily rejected islet allografts, and chronic hyperglycemia had no impact on the magnitude or quality of intragraft T-cell responses. In contrast, allospecific IgM and IgG were significantly decreased in Akita mice after alloimmunization. Thus, whereas diabetes influences host immune defense, hyperglycemia itself does not cause generalized alloimmune impairment. Our data suggest that immune compromise in diabetes due to hyperglycemia may not apply to cellular rejection of transplants.

Reversal of diabetes by transplantation of cryopreserved rat islets of Langerhans to the renal subcapsular space

Intraportal transplantation of cryopreserved islets of Langerhans has been shown to reverse streptozotocin-induced diabetes in rats. This study demonstrates the feasibility of transplanting 3,000 cryopreserved islets beneath the kidney capsule of syngeneic streptozotocin-diabetic Wistar-Furth rats. The clinical indices of plasma glucose, urine volume, and urine glucose returned to baseline values following implantation of fresh and cryopreserved islets. The clinical indices post-transplant and the K values (decline in plasma glucose concentration, percent/min) during intravenous glucose tolerance tests were not significantly different in rats receiving freshly isolated or cryopreserved islet grafts. The rapid return to the diabetic state after nephrectomy proved conclusively that the reversal of diabetes was due to the grafted islets. This was confirmed by immunocytochemical localization of well confirmed by immunocytochemical localization of well granulated islets beneath the kidney capsule. We conclude that cryopreserved islets reverse streptozotocin-induced diabetes after renal subcapsular implantation and that the clinical response is similar to fresh isografts.

IMPACT OF METABOLIC DEMAND ON ISLET TRANSPLANT SURVIVAL AND TOLERANCE INDUCTION: 3356

Introduction: While the roles of innate and adaptive immunity on allograft survival are well appreciated, the independent impact of metabolic demand on allograft survival and potential tolerance induction remains less clear. High metabolic demand on pancreatic islets is associated with intra-islet inflammation, especially as reported in Type 2 diabetes. Therefore, we determined if severe hyperglycemia impacts both human islet survival and the capacity to induce allograft tolerance in mouse models of diabetes. Methods and Results: In normal strains of mice, diabetes is commonly induced with the beta cell toxin streptozotocin (SZ). However, the degree of diabetes induced with SZ is notoriously variable and can sometimes recover over time. Therefore, we compared SZ-induced diabetic mice with spontaneously diabetic Ins-2akita (akita) recipients on the same C57Bl/6 (B6) genetic background for their relative propensity for islet allograft tolerance. Importantly, akita mice have an insulin secretory defect that results in a severe, irreversible form of diabetes. Daily hyperglycemia was consistently higher in diabetic B6 akita mice relative to corresponding SZ-induced diabetic B6 mice. Diabetec B6 akita mice accepted wild-type syngeneic B6 islet grafts for >100 days (12/12) without mononuclear cell infiltration, confirming that diabetes was not associated with autoimmunity. To compare tolerance induction in these two diabetic models, 400 allogeneic BALB/c islets were transplanted in these recipients with or without transient treatment with anti-CD154 therapy (hamster MR1, days -1, 2, 7,9 relative to transplant). Unmodified acute islet allograft rejection was significantly faster (p < .01) in diabetic B6 akita hosts (7.1 days) relative to SZ-induced diabetic B6 mice (13 days). Importantly, anti-CD154 therapy resulted in graft prolongation but not long term allograft acceptance in the majority of diabetic B6 akita recipients (only 1/14 allografts surviving >100 days). This was significantly different from results using SZ-induced diabetic B6 recipients in which 8/12 allografts survived > 100 day (p < .01). To test the impact of persistent metabolic demand on human islets in vivo, 1000-2000 human islets were grafted into immune-deficient B6 akita rag-/recipients. Interestingly, human islets that maintained euglycemia for greater than 30 days often spontaneously failed between 30-90 days with grafts displaying pronounced fibrosis and amyloid deposition. Conclusions: Results indicate that there can be significant differences in islet allograft survival depending on the model of diabetes examined (SZ-induced versus akita). Furthermore, prolonged severe metabolic demand can be sufficient to induce overt human islet graft failure over time independent of an adaptive immune response. Given the higher and persistent hyperglycemia found in akita recipients, we hypothesize that the degree of metabolic demand and/or tissue distress in islet transplants can be an independent variable in determining islet graft survival and can impede tolerance induciton.

Tolerance induction to pancreatic islet allografts

Purpose of reviewRecent clinical successes in pancreatic islet transplantation have spurred a resurgent interest in this approach to treating insulin-dependent diabetes. There is an ongoing need to develop therapies that will induce immunologic tolerance to islet transplants. The purpose of this review is to evaluate recent progress in promoting long-term allograft survival and tolerance in animal models of islet transplantation. Recent findingsAn increasing variety of cell surface molecules involved in costimulation, adhesion, and cytokine responses have proved useful as therapeutic targets for inducing indefinite islet allograft survival in non–autoimmune-prone rodent models. In some cases, treatment regimens can result in the generation of active, donor-specific regulatory tolerance. However, the primary application of islet transplantation is for the restoration of euglycemia in recipients with autoimmune (Type Ia) diabetes. Spontaneously autoimmune nonobese diabetic mice represent a formidable barrier to allograft tolerance relative to non–autoimmune-prone strains. Importantly, nonobese diabetic mice harbor inherent genetic resistance to allograft tolerance that can be dissociated from the autoimmune phenotype. Thus, the autoimmune-prone recipient represents a particularly stringent model for islet allograft tolerance. SummaryAlthough several approaches can induce tolerance induction to islet allografts in autoimmune-resistant strains of mice, relatively few therapies permit long-term islet allograft survival in nonobese diabetic or nonobese diabetic–related mouse strains. A primary future goal will be solving the inherent tolerance resistance in autoimmune-prone allograft recipients.