Conventional Immunosuppression is Compatible with Costimulation Blockade-Based, Mixed Chimerism Tolerance Induction

T-cell costimulatory blockade has emerged as an effective strategy to prevent allograft rejection in experimental models. We and others have reported that the beneficial effects of costimulation blockade can be negated when combined with certain immunosuppressants. The current study evaluates the compatibility of various immunosuppressive agents in a costimulation blockade-based, mixed chimerism tolerance protocol. The addition of conventional agents, including calcineurin inhibitors, did not interfere with tolerance induction. All mice developed multilineage macrochimerism and accepted donor allografts. Analysis of specific T-cell receptor utilization demonstrated selective deletion of donor-reactive T cells. Challenge with donor and third-party allografts confirmed donor-specific tolerance. Clinical introduction of costimulation blockade-based strategies will likely incorporate currently approved immunosuppressive agents. While it has been reported that certain conventional agents are detrimental to costimulation blockade-based strategies, our results suggest that these agents could safely be combined in clinical trials when used as part of a nonmyelosuppressive, mixed chimerism-based tolerance strategy.     

Bacterial infections, alloimmunity, and transplantation tolerance

Transplantation of solid organs across histocompatibility barriers in the absence of immunosuppression is invariably followed by acute allograft rejection. Although several immunosuppressive regimens have been developed to prevent allograft rejection, these global immunosuppressive agents effectively inhibit all T cells, leaving the host vulnerable to infections. Thus, a major goal in transplantation immunology is to induce donor-specific tolerance that results in the extended suppression of allograft-specific immune responses, while leaving the remainder of the immune system competent to fight infections and malignancies. Initial successes in identifying approaches that successfully induce transplantation tolerance in experimental models have led to a newer research focus of identifying potential barriers to the induction of such tolerance as well as events that may reverse established allograft tolerance. Both clinical and experimental studies have identified bacterial infections as a possible trigger of allograft rejection. Recently, experimental models of transplantation tolerance have identified that bacterial signals can promote acute allograft rejection either by preventing the induction of transplantation tolerance or by reversing tolerance after it has been stably established. This review summarizes experimental and clinical literature supporting the hypothesis that bacterial infections and innate immunity can qualitatively and quantitatively alter adaptive alloreactivity through effects on innate immune responses.     

CD8+ T‐Cell Depletion and Rapamycin Synergize with Combined Coreceptor/Stimulation Blockade to Induce Robust Limb Allograft Tolerance in Mice

The growing development of composite tissue allografts (CTA) highlights the need for tolerance induction protocols. Herein, we developed a mouse model of heterotopic limb allograft in a stringent strain combination in which potentially tolerogenic strategies were tested taking advantage of donor stem cells in the grafted limb. BALB/c allografts were transplanted into C57BL/6 mice treated with anti‐CD154 mAb, nondepleting anti‐CD4 combined to either depleting or nondepleting anti‐CD8 mAbs. Some groups received additional rapamycin. Both depleting and nondepleting mAb combinations without rapamycin only delayed limb allograft rejection, whereas the addition of rapamycin induced long‐term allograft survival in both combinations. Nevertheless, robust donor‐specific tolerance, defined by the acceptance of a fresh donor‐type skin allograft and simultaneous rejection of third‐party grafts, required initial CD8⁺ T‐cell depletion. Mixed donor‐recipient chimerism was observed in lymphoid organs and recipient bone marrow of tolerant but not rejecting animals. Tolerance specificity was confirmed by the inability to produce IL‐2, IFN‐γ and TNF‐α in MLC with donor antigen while significant alloreactivity persisted against third‐ party alloantigens. Collectively, these results show that robust CTA tolerance and mixed donor‐recipient chimerism can be achieved in response to the synergizing combination of rapamycin, transient CD8⁺ T‐cell depletion and costimulation/coreceptor blockade.     

From current immunosuppressive strategies to clinical tolerance of allografts

In order to prevent allograft rejection, most current immunosuppressive drugs nonspecifically target T-cell activation, clonal expansion or differentiation into effector cells. Experimental models have shown that it is possible to exploit the central and peripheral mechanisms that normally maintain immune homeostasis and tolerance to self-antigens, in order to induce tolerance to alloantigens. Central tolerance results from intrathymic deletion of T cells with high avidity for thymically expressed antigens. Peripheral tolerance to nonself-molecules can be achieved by various mechanisms including deletion of activated/effector T cells, anergy induction and active regulation of effector T cells. In this article, we briefly discuss the pathways of allorecognition and their relevance to current immunosuppressive strategies and to the induction of transplantation tolerance (through haematopoietic mixed chimerism, depleting protocols, costimulatory blockade and regulatory T cells). We then review the prospect of clinical applicability of these protocols in solid organ transplantation.     

Comparison of chimeric acid and non-chimeric tolerance using posttransplant total lymphoid irradiation: cytokine expression and chronic rejection.

BACKGROUND: Previous studies showed that an intravenous infusion of donor blood cells facilitates tolerance to ACI heart allografts in Lewis rat hosts given posttransplant total lymphoid irradiation (TLI) and anti-thymocyte globulin (ATG). The object of the current study was to compare tolerance induction using donor cells that do or do not induce chimerism. METHODS: Normal peripheral blood mononuclear cells (PBMC), granulocyte colony-stimulating factor (G-CSF)-mobilized PBMC, and bone marrow (BM) cells from ACI donors were tested for their capacity to prolong ACI heart allograft survival in Lewis hosts. Chimerism, anti-donor cell reactivity, and cytokine gene expression in grafts were determined. RESULTS: Intravenous injections of equal numbers of all three donor cells markedly prolonged graft survival (median: >164 to >175 days) as compared to uninjected controls (median: 53 days). Chimerism among T and B cells in the blood was determined by immunofluorescent staining in hosts bearing long-term (> 150 days) grafts. Although no chimerism was detected in hosts given normal or G-CSF-mobilized PBMC, chimerism was detected at variable levels in all hosts given BM cells. Vigorous anti-donor reactivity in the mixed leukocyte reaction was present only in non-chimeric hosts. Long-term grafts from hosts given normal ACI PBMC developed chronic rejection, but those from hosts given ACI BM cells did not. The latter hosts showed the lowest levels of intragraft cytokine mRNA. CONCLUSIONS: Chimeric tolerance is more robust than non-chimeric tolerance in the model of posttransplant TLI, ATG, and donor cell infusion, and is associated with less chronic rejection.     

Clinical experience with mixed chimerism to induce transplantation tolerance

Lymphohematopoietic chimerism was first shown to be associated with donor‐specific allograft tolerance more than 60 years ago. However, early clinical experience with bone marrow transplantation soon revealed that conventional, myeloablative approaches were far too toxic and the risk of graft‐versus‐host disease too great to justify using this technology for the purpose of organ allograft tolerance induction in the absence of malignant disease. In this review, we discuss a step‐wise approach that has been applied by several centers to establish less toxic approaches to using hematopoietic cell transplantation (HCT) for tolerance induction. These steps include (i) feasibility and efficacy data for tolerance induction in large animal models; (ii) safety data in clinical trials for patients with hematologic malignancies; and (iii) pilot trials of combined HCT and kidney transplantation for tolerance induction. Thus far, only one published trial conducted at the Massachusetts General Hospital in Boston has achieved long‐term acceptance of human leukocyte antigen‐mismatched kidney allografts without chronic immunosuppressive therapy. Alternative protocols have been successful in large animals, but long‐term organ allograft tolerance has not been reported in patients. Thus, proof‐of‐principle that nonmyeloablative induction of mixed chimerism can be used intentionally to induce organ allograft tolerance has now been achieved. Directions for further research to make this approach applicable for a broader patient population are discussed.     

Prolonged limb allograft survival with CD 40 costimulation blockade, T-cell depletion, and megadose donor bone-marrow transfusion

The purpose of this study was to determine the efficacy of a treatment regimen consisting of CD 40 costimulation blockade, T-cell depletion, and megadose donor bone marrow transfusion in the limb allograft model. C57Bl/6 mice underwent limb transplantation from Balb/c mice and received MR1 (anti-CD 40 ligand monoclonal antibody), and CD4(+) and CD8(+) T cell-depleting antibodies with and without 120 x 10(6) donor bone-marrow transfusion. Recipients treated only with antibodies showed rejection at 51.4+/-17 (mean+/-SEM) days, while those who also received donor bone marrow had allograft survival of 67+/-16.4 days, with a range up to 91 days. Treated specimens with rejection had less lymphocytic infiltration than untreated controls. Recipients of donor bone marrow also demonstrated early mixed chimerism, which disappeared after 1 month. While allograft survival was prolonged, tolerance was not achieved, and the mechanism of rejection was more consistent with a chronic process.     

Chimerism,Graft Survival,and Withdrawal of Immunosuppressive Drugs in HLA Matched and Mismatched Patients After Living Donor Kidney and Hematopoietic Cell Transplantation

Thirty‐eight HLA matched and mismatched patients given combined living donor kidney and enriched CD34⁺ hematopoietic cell transplants were enrolled in tolerance protocols using posttransplant conditioning with total lymphoid irradiation and anti‐thymocyte globulin. Persistent chimerism for at least 6 months was associated with successful complete withdrawal of immunosuppressive drugs in 16 of 22 matched patients without rejection episodes or kidney disease recurrence with up to 5 years follow up thereafter. One patient is in the midst of withdrawal and five are on maintenance drugs. Persistent mixed chimerism was achieved in some haplotype matched patients for at least 12 months by increasing the dose of T cells and CD34⁺ cells infused as compared to matched recipients in a dose escalation study. Success of drug withdrawal in chimeric mismatched patients remains to be determined. None of the 38 patients had kidney graft loss or graft versus host disease with up to 14 years of observation. In conclusion, complete immunosuppressive drug withdrawal could be achieved thus far with the tolerance induction regimen in HLA matched patients with uniform long‐term graft survival in all patients.     

Transplantation of a whole ear allograft by immunological induction of donor-specific tolerance by bone marrow transplantation: An experimental study in rabbits

Previous studies in our laboratory have shown that giving bone marrow cells through the portal vein or intraosseous route is likely to be beneficial to tolerance of induction of allografts in rabbits. Using this model, we tested whether a less severe regimen for conditioning of the host can prevent rejection of allografts. Rabbits were given a single intraosseous injection of donor bone marrow cells and total body irradiation (7 Gy) and transplantation of skin and ear allografts. Mean skin allograft survival for this treatment was 88 days, which was similar to the results of our earlier study. A donor ear was accepted for more than a year with no signs of rejection. These results suggest that a single intraosseous injection of donor bone marrow cells is sufficient for induction of donor-specific tolerance in rabbits and that immunosuppressive agents may not be needed.     

Transplantation of a whole ear allograft by immunological induction of donor-specific tolerance by bone marrow transplantation: an experimental study in rabbits.

Previous studies in our laboratory have shown that giving bone marrow cells through the portal vein or intraosseous route is likely to be beneficial to tolerance of induction of allografts in rabbits. Using this model, we tested whether a less severe regimen for conditioning of the host can prevent rejection of allografts. Rabbits were given a single intraosseous injection of donor bone marrow cells and total body irradiation (7 Gy) and transplantation of skin and ear allografts. Mean skin allograft survival for this treatment was 88 days, which was similar to the results of our earlier study. A donor ear was accepted for more than a year with no signs of rejection. These results suggest that a single intraosseous injection of donor bone marrow cells is sufficient for induction of donor-specific tolerance in rabbits and that immunosuppressive agents may not be needed.     

Avenues for immunomodulation and graft protection by gene therapy in transplantation

Organ transplantation represents the only definitive therapy for many causes of end-organ failure. However, the universal success of this therapy is limited by chronic allograft rejection, the side effects of chronic immunosuppressive therapy, and a severe shortage of donor organs. Presently, the success of solid-organ transplantation depends on the continuous administration of toxic and nonspecific immunosuppressive agents, therapies that present risks for opportunistic infection, malignancy, and a variety of agent-specific side effects. To promote the use of transplantation with limited risk of long-term sequelae, three dominant research challenges emerge: (i) elimination of the need for exogenous immunosuppression by immunological tolerance induction; (ii) prevention of chronic rejection/graft dysfunction; and (iii) expansion of available organs for transplantation. Gene therapy may provide significant advances and solutions in each of these areas. Rejection of the graft in the immediate post-transplant period has been attacked through the transfer of immunomodulatory molecules in addition to tolerance inducing approaches. Chronic graft rejection may be similarly addressed through permanent tolerance induction or alternatively through the introduction of molecules to resist chronic graft damage. Genetic manipulation of stem cells may ultimately produce transgenic animals to serve as tissue donors to overcome the limited donor organ supply. This review will highlight ongoing developments in the translation of gene therapy approaches to the challenges inherent in transplantation.     

Approaches to transplantation tolerance in humans

Although transplantation tolerance to organ allografts has been achieved using a wide variety of immunologic interventions in laboratory animals, few tolerance induction protocols with complete immunosuppressive drug withdrawal have been tested in humans. Preclinical and clinical studies of the use of total lymphoid irradiation for the induction of chimeric and nonchimeric tolerance are summarized here.     

Current status and prospect of T cell costimulatory blockade in transplantation

Organ transplantation has been widely accepted as a routine medical treatment in Japan. The recent introduction of new immunosuppressive reagents may improve outcome after transplantation. However, further investigations are required to achieve the prevention of chronic rejection and the induction of donor-specific tolerance in clinical transplantation. The potential of costimulatory blockade in controlling allograft rejection is now extensively under investigation. Although costimulatory blockade can prevent rejection and induce tolerance experimentally, there are several issues that need to be clarified before its clinical application. Recently, several novel costimulatory pathways have also been reported. Each costimulatory pathway has distinct and unique immunological roles in T cell activation and down-regulation. Upon the perception of underlying mechanisms, the theoretical and effective use of costimulatory blockade in combination with conventional immunosuppression and/or novel molecular targeting therapies might make a major breakthrough in clinical transplantation.     

Coronary atherosclerosis in cardiac transplant patients treated with total lymphoid irradiation.

BACKGROUND: Multiple episodes of rejection following cardiac transplantation have been associated with an increased incidence of coronary atherosclerosis. Total lymphoid irradiation (TLI) has been shown to be a successful treatment for persistent allograft rejection, but its effect on coronary arterial disease has yet to be evaluated. METHODS: From 1987 to 1999, 40 patients required TLI for persistent or recurrent allograft rejection following heart transplantation. Each patient's (Group 1, n = 31) post-transplant coronary angiograms were examined and compared with those of a control group (Group 2, (n = 32) matched for time of transplantation. Degree of coronary stenosis was assessed on a 6-point scale. All patients received induction therapy (rabbit anti-thymocyte globulin or OKT3) and standard triple immunosuppressive therapy. TLI (80 cGy x 10 fractions) was used for the treatment of recurrent or persistent rejection on the basis of clinical indications. Actuarial survival, number and treatment of rejection episodes, and severity of coronary artery disease were compared in each group. RESULTS: Recipient gender, age, race and cytomegalovirus (CMV) status at time of transplant, along with donor gender, CMV status and graft ischemia time, were similar in both groups. Group 1 donor age was younger than that of Group 2 (22.2 +/- 11.2 vs 31.5 +/- 13.6 years, p = 0.004), and the indication for surgery in Group 1 patients was more likely to be ischemic heart disease (15 of 31 vs 6 of 32, p = 0.02). Mean follow-up was 5.7 +/- 3.5 years in Group 1 vs 6.9 +/- 3.8 in Group 2 (p = NS). Group 1 had more rejection episodes (4.4 +/- 2.2 vs 2.3 +/- 2.0, p = 0.0002) and more steroid treatments (9.78 +/- 4.0 g vs 5.14 +/- 4.7 g, p < 0.0001), but less coronary artery disease compared with Group 2 (p = 0.035). CONCLUSIONS: Despite multiple episodes of rejection, patients treated with TLI after cardiac transplant appear to develop less coronary atherosclerosis than appropriately matched controls.     

Tracking of TCR‐Transgenic T Cells Reveals That Multiple Mechanisms Maintain Cardiac Transplant Tolerance in Mice

Solid organ transplantation tolerance can be achieved following select transient immunosuppressive regimens that result in long‐lasting restraint of alloimmunity without affecting responses to other antigens. Transplantation tolerance has been observed in animal models following costimulation or coreceptor blockade therapies, and in a subset of patients through induction protocols that include donor bone marrow transplantation, or following withdrawal of immunosuppression. Previous data from our lab and others have shown that proinflammatory interventions that successfully prevent the induction of transplantation tolerance in mice often fail to break tolerance once it has been stably established. This suggests that established tolerance acquires resilience to proinflammatory insults, and prompted us to investigate the mechanisms that maintain a stable state of robust tolerance. Our results demonstrate that only a triple intervention of depleting CD25⁺ regulatory T cells (Tregs), blocking programmed death ligand‐1 (PD‐L1) signals, and transferring low numbers of alloreactive T cells was sufficient to break established tolerance. We infer from these observations that Tregs and PD‐1/PD‐L1 signals cooperate to preserve a low alloreactive T cell frequency to maintain tolerance. Thus, therapeutic protocols designed to induce multiple parallel mechanisms of peripheral tolerance may be necessary to achieve robust transplantation tolerance capable of maintaining one allograft for life in the clinic.     

Contemporary immunosuppression in renal transplantation.

Over the past 3 decades, renal allograft survival has improved significantly as a result of the development of powerful immunosuppressive agents. Nevertheless, the overall half-life of renal allografts has increased marginally during that time period, owing to drug-related nephrotoxicity and chronic rejection. New immunosuppressive agents are being evaluated because of the need for a reduction in the dose of nephrotoxic calcineurin inhibitors and corticosteroids. Additional agents have demonstrated the ability to retard the onset of chronic rejection in preclinical transplant models. In concert with these efforts, approaches are in development to alleviate the ever increasing shortage of donor organs, including the as yet unrealized goals of successful and practical xenotransplantation and the bioartificial kidney. Further identification and development of novel agents that target the specific components of the allograft response will provide the key to the achievement of donor-specific tolerance, the "Holy Grail" of solid organ transplantation.     

A novel mechanism of action for anti-thymocyte globulin: induction of CD4+CD25+Foxp3+ regulatory T cells

T cell-depleting agents are being tested as part of clinical tolerance strategies in humans with autoimmunity and transplantation. The immunosuppressive activity of anti-thymocyte globulin (ATG) has been thought to result primarily from depletion of peripheral lymphocytes. Herein is reported for the first time that ATG but not anti-CD52 mAb (alemtuzumab) or the IL-2R antagonists causes rapid and sustained expansion of CD4+CD25+ T cells when cultured with human peripheral blood lymphocytes. These cells display enhanced expression of the regulatory markers glucocorticoid-induced TNF receptor, cytotoxic T lymphocyte-associated antigen-4 (CTLA-4), and forkhead box P3 and efficiently suppress a direct alloimmune response of the original responder lymphocytes. It is interesting that the cells do not suppress memory responses to the recall antigen mumps. Ex vivo expansion of regulatory T cells is due mainly to conversion of CD4+CD25- into CD4+CD25+ T cells and to a lesser degree to proliferation of natural CD4+CD25+ T cells. The induction of regulatory T cells depends on production of Th2 cytokines in the generating cultures. These novel data suggest that ATG not only may promote expansion/generation of regulatory T cells but also may be useful in future ex vivo expansion of these cells for cellular therapy in autoimmunity and clinical transplantation.     

Establishment of fully xenogeneic (mouse-->rat) bone marrow chimeras: evidence for normal development and clonal deletion of mouse T cells

BACKGROUND: Xenotransplantation is a potential solution to the critical shortage of transplantable organs. However, conventional immunosuppressive agents do not control the vigorous cellular and humoral rejection across species disparities. The induction of donor specific tolerance via bone marrow chimerism may be a method to avoid xenograft rejection. In xenogeneic chimeras, T cell repertoire selection plays an important role in the induction of tolerance. Until now a model of mouse-->rat multilineage chimerism has not been reported. This study reports the establishment of fully xenogeneic mouse-->rat multilineage chimeras and evaluates the role of T cell development and repertoire selection in tolerance induction in a xenogeneic environment. METHODS: Recipient rats were irradiated at a dose of total body irradiation ranging between 800-1100 cGy and injected with 120-300x10(6) donor mouse bone marrow cells. Chimeras were typed for engraftment at 4 weeks and then monthly thereafter. T cell repertoire was evaluated in chimeras using two-color flow cytometry and monoclonal antibodies directed against the variable portion of the beta chain of the T cell receptor. RESULTS: Fully xenogeneic multilineage bone marrow chimerism was produced in a mouse-->rat model by using ablative radiation and a high dose of donor cells. Mouse T cells develop in a phenotypically normal fashion in chimeric rats and the host rat is capable of deleting T cells that are reactive to the donor mouse strain. CONCLUSION: Long-term multilineage bone marrow chimerism can be produced in a mouse-->rat bone marrow transplant model. Mouse T cells develop in a phenotypically normal fashion and negative selection of specific T cell receptor-Vbeta occurs in a xenogeneic environment in a predictable fashion paralleling that for syngeneic or allogeneic transplantation.     

Effects of Cyclosporine on Transplant Tolerance: The Role of IL-2

Allograft(dagger) transplant outcome, rejection or tolerance, depends upon striking a balance between the pertinent cytopathic and regulatory T cells. The drug cyclosporine is a widely used immunosuppressive agent among transplant recipients. Previous studies have demonstrated that cyclosporine blocks apoptosis of activated T cells and the ability of costimulation blockade based regimens to create peripheral transplant tolerance. We now test the hypothesis that the mechanism by which cyclosporine blocks tolerance induction is IL-2 dependent, and linked to a detrimental effect upon T(reg) function. Our study demonstrates that cyclosporine blocks IL-2 gene expression and activation induced cell death (AICD) of alloreactive T effector cells. We also show that cyclosporine abolishes the beneficial effects of a donor specific transfusion (DST) plus anti-CD154 monoclonal antibody (alpha CD154) regimen on enhanced T(regs) function and allograft tolerance induction. Interestingly, provision of IL-2/Fc, a long-lived form of IL-2, completely reverses the detrimental effects of this adjunctive cyclosporine treatment on AICD of alloreactive T effectors, T(regs) function and tolerance induction. Furthermore, in a MHC mismatched islet allograft model, the combination of cyclosporine with IL-2/Fc permitted long-term allograft survival and induced alloantigen specific allograft tolerance. The combination of IL-2/Fc and cyclosporine treatment may provide a new clinical strategy to promote transplant tolerance.