Induction Of Allograft Tolerance Research Articles

Pancreatic islet xenotransplantation: barriers and prospects.

Dramatic clinical advances indicate that pancreatic islet transplants can reliably restore euglycemia in insulin-dependent patients. However, clinical success actually highlights the pronounced deficiency of allogeneic pancreata available for islet isolation. This pressing issue has revitalized ongoing efforts to develop surrogate donor sources. Xenogeneic donors form a potential alternative tissue source because they can be generated in large numbers and are amenable to genetic engineering. However, there is less understanding of the innate and adaptive immune barriers to islet xenografts relative to those encountered by allografts. Presented evidence indicates that both innate and antigen-specific adaptive immune responses significantly contribute to islet xenograft rejection. Recent evidence suggests that the capacity to induce tolerance to islet xenografts may not differ markedly from strategies used to induce allograft tolerance.

CTLA-4. FasL induces alloantigen-specific hyporesponsiveness.

The APC:T cell interface can be effectively targeted with immunotherapeutic proteins. We previously described a unique trans signal converter protein, CTLA-4. Fas ligand (FasL), that has the inherent capacities to tether the T cell inhibitor FasL (CD95 ligand) to the surfaces of B7 (CD80 and CD86)-positive APC (via CTLA-4:B7 interaction), and in so doing, to simultaneously interfere with B7-to-CD28 T cell activation signals. Given the continuing need for agents capable of inducing allograft tolerance without generalized immunosuppression, we have explored in depth the functional activity of CTLA-4. FasL in human allogeneic MLR. CTLA-4. FasL inhibits 1 degrees MLR and induces specific hyporesponsiveness in 2 degrees MLR, with both effects only partially reversible with exogenous IL-2. Moreover, the presence of exogenous IL-2 during the 1 degrees MLR does not affect the induction of hyporesponsiveness upon restimulation. Furthermore, CTLA-4. FasL enables partial activation of allostimulated T cells, reduces the fraction of actively dividing cells, and increases the percentage of dead cells among dividing T cells. Taken together, these findings suggest that CTLA-4. FasL-mediated inhibition of secondary alloantigenic responses involves both anergy induction and clonal deletion. Thus, CTLA-4. FasL, a paradigmatic trans signal converter protein, manifests unique functional properties and emerges as a potentially useful immunotherapeutic for modulating alloresponsiveness.

Induction of allograft tolerance through costimulatory blockade: first selection of drugs in vitro

The development of an in vitro assay predicting the chances of graft survival after treatment with immunoregulatory agents is a major topic in transplantation. Antibodies (Abs) interfering in the costimulatory pathway are promising candidates for the induction of tolerance. To evaluate these antibodies for clinical use studies non-human primates are the only feasible option due to species specificity of the antibodies. Peripheral blood mononuclear cells, isolated from a large panel of rhesus monkeys, were used in a unidirectional mixed lymphocyte reaction to evaluate the ability of antibodies blocking the costimulatory pathway, to affect both primary and secondary proliferative and cytolytic allospecific immune responses in vitro. These blocking antibodies were also used in protocols prolonging allograft survival in a life-supporting kidney allotransplant model in rhesus macaques. The ultimate aim is to establish a correlation between parameters obtained in vitro and the success of transplantation in vivo. The combination of anti-CD80 and anti-CD86 resulted in a complete abrogation of the primary alloresponse as measured in a proliferation assay. Adding anti-CD40 significantly reduced this inhibitory effect although the in vivo effects of this antibody have been shown to be beneficial. The secondary response was most prominently inhibited by the combination of anti-CD80/86. Paradoxically, anti-CD40 alone markedly inhibited the secondary proliferative response, but did not add to the inhibitory effect of the combination of anti-CD80/86. The cytolytic response was inhibited maximally only when CsA was added to the combination of anti-CD80/86. Treatment with monoclonal antibodies alone without immunosuppressive drugs was sufficient to maintain graft survival during the time of treatment in most animals. However, rejection was initiated as soon as the treatment ceased and no tolerance, resulting in long-term graft and patient survival, was established. The complete inhibition of primary alloresponses and the partial inhibition of secondary proliferative alloresponses correlate with prolonged graft survival during treatment, but have no predictive value for the success of tolerance induction for kidney allografts in rhesus monkeys.

Prevention of kidney allograft rejection using anti-CD40 and anti-CD86 in primates.

Costimulation blockade has been proposed to induce allograft tolerance. We combined an antagonist anti-CD40 monoclonal antibody (mAb) with an antagonist anti-CD86 mAb in a rhesus monkey kidney allograft model. We chose this combination because it leaves CD80-CD152 signaling unimpaired, allowing for the down-regulatory effect of CD152 signaling to take place through this pathway. Rhesus monkeys underwent transplantation with a major histocompatibility complex-mismatched kidney. One group of animals received anti-CD40 alone, and a second group received the combination of anti-CD40 and anti-CD86, twice weekly for 56 days. Three animals with low levels of anti-CD40 rejected the transplanted kidney while still receiving treatment. Three animals with high levels of anti-CD40 rejected at days 91, 134, and 217 with signs of chronic rejection. Animals treated with the combination of anti-CD40 and anti-CD86 mAbs rejected their kidneys at days 61, 75, and 78, shortly after cessation of treatment. Two animals were killed on days 71 and 116 with a blocked ureter. These animals developed virtually no signs of tubulitis or infiltration during treatment and no donor-specific alloantibodies. Both treatment protocols prevented rejection for the duration of the treatment in most animals. Blocking costimulation by anti-CD40 or by anti-CD40 plus anti-CD86 may be an effective method to prevent graft rejection and may obviate the need for other immunosuppressive drugs, especially in the immediate posttransplantation period.

T-cell growth factors and the allograft response

Allograft rejection-like tolerance is a T-cell–mediated event. Acquisition of transplant tolerance, however, is a learned process, and T cells need to engage actively in alloantigens to learn to tolerate the graft. T-cell growth factors play an important role in regulating multiple aspects of the allograft response. It is true that T-cell growth factors can drive clonal expansion and functional maturation of alloreactive T cells; therefore, targeting growth factors and their receptor components remains one of the key aspects in transplantation. There is compelling evidence that growth factors also regulate the induction of peripheral allograft tolerance through programming activated T cells for apoptosis and guiding the development of regulatory T cells. Thus, understanding precisely the role of T-cell growth factors in regulating T-cell homeostasis and in developing active immune regulation is critically important in transplant tolerance.

Tolerance induction in rats, using a combination of anti-CD154 and donor splenocytes, given once on the day of transplantation.

Donor-specific tolerance induction remains an attractive objective that generates much research in the field of transplantation. Unfortunately, most of the protocols available involve pregraft conditioning, making these treatments incompatible with clinical applications. LEW.1A rats were grafted with histoincompatible LEW.1W hearts. On the day of transplantation, recipients were treated with anti-CD40L combined with donor splenocytes. The hearts were evaluated for graft survival; cellular infiltrate and intragraft cytokines were determined using real-time reverse transcriptase-polymerase chain reaction. Tolerance induction was assessed by skin grafting and adoptive transfers. The combination of a single injection of anti-CD40L and donor splenocytes, given on the day of surgery, allowed 40% of cardiac allografts to survive long-term (mean survival time=66.3 day). The cellular composition or the extent of graft infiltrate was not modified but was associated with a massive decrease of proinflammatory cytokines expression within the graft. Long-term survivors accepted donor-matched skin grafts, and leukocytes harvested from these animals transferred tolerance into irradiated freshly grafted recipients. A combination of costimulation blockade and donor cells, given once at the time of transplantation, is sufficient to induce allograft tolerance in rats.

Long-Term Immunologic Induction of Donor-Specific Tolerance to Skin Allografts by Bone Marrow Transplant in Rabbits

The induction of donor-specific tolerance to skin allografts was investigated in rabbits using bone marrow transplantation techniques reported to be effective in mice. Various routes of bone marrow transplantation (i.e., intravenous, portal venous, or intraosseous) were also examined. In regimen A, the animals were treated with portal venous injection of bone marrow cells from the donor on day 0 and intravenous injection of bone marrow cells from the same donor on posttransplant day 5. In regimen B, the animals were treated with portal venous and intraosseous injections of donor bone marrow cells on day 0 and intravenous injection of bone marrow cells from the same donor on posttransplant day 5. In regimen C, the animals were given intraosseous injection of donor bone marrow cells on day 0 and intravenous injection of bone marrow cells from the same donor on posttransplant day 5. It was found that regimens B and C were more effective than regimen A in prolonging allograft survival. The results demonstrate that induction of allograft tolerance can be achieved by bone marrow transplantation in a rabbit model. This protocol deserves further study in other large animal models. (Plast. Reconstr. Surg. 111: 291, 2003.)

Long-term immunologic induction of donor-specific tolerance to skin allografts by bone marrow transplant in rabbits.

The induction of donor-specific tolerance to skin allografts was investigated in rabbits using bone marrow transplantation techniques reported to be effective in mice. Various routes of bone marrow transplantation (i.e., intravenous, portal venous, or intraosseous) were also examined. In regimen A, the animals were treated with portal venous injection of bone marrow cells from the donor on day 0 and intravenous injection of bone marrow cells from the same donor on posttransplant day 5. In regimen B, the animals were treated with portal venous and intraosseous injections of donor bone marrow cells on day 0 and intravenous injection of bone marrow cells from the same donor on posttransplant day 5. In regimen C, the animals were given intraosseous injection of donor bone marrow cells on day 0 and intravenous injection of bone marrow cells from the same donor on posttransplant day 5. It was found that regimens B and C were more effective than regimen A in prolonging allograft survival. The results demonstrate that induction of allograft tolerance can be achieved by bone marrow transplantation in a rabbit model. This protocol deserves further study in other large animal models.

T-cell depleting antibodies: new hope for induction of allograft tolerance in bone marrow transplantation?

Graft versus host disease (GVHD) remains the main barrier to successful allogeneic bone marrow transplant outcomes. Depletion of graft T cells is an effective way of reducing the incidence of acute and chronic GVHD, and a variety of methods have been used to achieve this depletion. Donor CD8+ T cells seem to be the critical effector cells; GVHD is reduced when the depletion process eliminates these cells, but not when CD4 cells are targeted alone. However, despite the successful reduction in GVHD, transplant outcomes are usually inferior with T-cell depleted transplants, because of increased graft failure, infections and relapse. Alternative approaches are needed. In vivo T-cell depletion, using antithymocyte globulin (ATG) as part of the conditioning regimen, seems an attractive option. Pre-transplant ATG lingers in the bone marrow to deplete engrafting donor T cells, but also depletes host T cells to prevent graft rejection and allow de-escalation of the conditioning regimen. It also avoids the need for graft manipulation with its associated costs, need for expertise and CD34+ cell loss. The efficacy of pre-transplant horse ATG remains anecdotal but it has been reported to modestly lower GVHD in single arm studies. Rabbit ATG has been studied in prospective randomised trials. There is evidence of a dose-response effect in reducing GVHD; however, there was no improvement in outcome, because of increased mortality associated with infection. In contrast, pre-transplant alemtuzumab (campath-1H) or an earlier version of this molecule (campath-1G), which target CD52+ cells, do appear to be effective in reducing both acute and chronic GVHD. There is speculation that this is not solely due to the effect of campath on T cells but that it may also be due to the elimination of host antigen-presenting cells (APC), which seem to be important in GVHD pathogenesis. Host APC are more efficient at expressing endogenous and exogenous host antigens on class I MHC to donor CD8+ cells than donor APC, which need to cross-prime exogenous antigen. Campath-1G eliminates host dendritic cells by the time of graft infusion, supporting this as a possible mechanism of action. Pre-transplant alemtuzumab has not yet been studied in a prospective randomised study, and this is required to quantify any benefit on outcome; despite this, published studies do show cause for optimism.

Study of the effect of indoleamine 2,3-dioxygenase on murine mixed lymphocyte reactions and skin allograft rejection

ACUTE REJECTION of allografts is mediated by the coordinated infiltration and effector functions of alloantigen-specific T cells. After infiltration of transplanted allografts, host T cells are activated to produce proinflammatory cytokines and to express cytolytic activities that mediate destruction of the graft tissue. It is known that interferon(INF) is often produced and detected in allografts during rejection. Although INFup-regulates MHC molecule expression and enhances alloantigen presentation in vitro, it is not required for acute rejection of fully allogeneic (MHC class I–disparate and MHC class II–disparate) grafts. Instead, INFhas an essential immunoregulatory function in vivo. INFcan facilitate the induction of long-term allograft survival and tolerance to protein antigens partly by limiting the proliferation of activated T lymphocytes. Indoleamine 2,3-dioxygenase (IDO) is the first and rate-limiting enzyme in the L-tryptophan-kynurenine pathway that converts the essential amino acid, Ltryptophan (L-TRP), to N-formylkynurenine in mammalian extrahepatic tissues. It recently was proposed that IDO-mediated immunosuppression might prevent rejection of the fetus by the maternal T-cell response. Although several studies have circumstantially linked IDO induction to certain anti-proliferative and antimicrobial effects of immune stimuli, different and potentially detrimental consequences of IDO induction also have been proposed. Although the mechanism of IDO induction in vivo is unclear, it is widely accepted that IDO is up-regulated by INF– dependent and INF– independent mechanisms, and down-regulated by interleukin (IL)-4 and TGF. It has not yet been established, however, whether the physiological role of IDO regulation by these cytokines is beneficial or detrimental, nor whether this enzyme has an effect on allograft rejection. Several studies suggest that INFinduction of IDO exerts anti-microbial and anti-proliferative effects, possibly by depleting L-TRP. By treating skin allograft mice with 1-methyl-DL-tryptophan, which competes with L-TRP for IDO, we tested whether IDO might play an important role in the skin allograft rejection as a mechanism for regulating T-cell responses after producing INF. MATERIALS AND METHODS Mice BALB/c (H-2), C57BL/6 (H-2), B6.C-H2bm1, and B6.C-H2bm12 female mice, aged 6 to 8 weeks, were purchased from the Animal Production Area, NCI-Frederick (Frederick, Md, USA).

Rhesus monocyte-derived dendritic cells modified to over-express TGF-??1 exhibit potent veto activity1

The tolerogenic activity of allogeneic bone marrow cells (BMCs) associates with functional inactivation of alloreactive T cells and has been attributed to a veto effect. Studies in mice and rhesus monkeys indicated that the CD8alpha molecule expressed on a subpopulation of allogeneic BMCs is necessary to induce signal transduction within the BMCs to increase veto effector molecules such as transforming growth factor (TGF)-beta1. In vitro activation of alloreactive cytotoxic T-lymphocyte precursor enhances their susceptibility to veto-mediated functional inactivation by specific alloantigen-bearing BMCs. Accordingly, we examined a hypothesis that mature rhesus monkey (Rh) monocyte-derived dendritic cells (MDDCs) modified by gene transfer to over-express active TGF-beta1 might mediate veto activity without the need to express CD8alpha. Rh MDDCs were modified by recombinant adenovirus (Ad) transduction and characterized by phenotype and functional studies. Rh MDDC transduction with Ad vectors using conventional methods was remarkably inefficient. However, a single-chain anti-CD40/soluble Coxsackie and adenovirus receptor-fusion protein (G28/sCAR) permitted high-efficiency transduction of Rh MDDCs by retargeting Ad to Rh MDDC CD40. Mature Rh MDDCs that were transduced to overexpress active TGF-beta1 (AdTGF-beta1 Rh MDDC) significantly suppressed alloimmune responses in [ H]thymidine uptake mixed leukocyte reaction assays. We showed by the carboxyfluorescein succinimidyl ester dilution method that allogeneic mature AdTGF-beta1 Rh MDDCs inhibited proliferation of CD4 and CD8 responder T cells. Notably, AdTGF-beta1 Rh MDDC abrogated alloimmune responses induced by control AdGFP Rh MDDC in an antigen-specific manner. These results suggest that nonhuman primate mature MDDCs can be genetically engineered to function as alloantigen-specific cellular immunosuppressants, an approach that has potential to facilitate induction of allograft tolerance in vivo.

Role for thymic and splenic regulatory CD4+ T cells induced by donor dendritic cells in allograft tolerance by LF15-0195 treatment.

A 20-day treatment with LF15-0195, a deoxyspergualine analogue, induced allograft tolerance in a fully MHC-mismatched heart allograft model in the rat. Long-term allografts displayed minimal cell infiltration with no signs of chronic rejection. CD4+ spleen T cells from tolerant LF15-0195-treated recipients were able to suppress in vitro proliferation of allogeneic CD4+ T cells and to transfer tolerance to second syngeneic recipients, demonstrating dominant suppression by regulatory cells. A significant increase in the percentage of CD4+CD25+ T cells was observed in the thymus and spleen from tolerant LF15-0195-treated recipient. In vitro direct stimulation with donor APCs demonstrated that CD4+ regulatory T cells proliferated weakly and expressed low levels of IFN-gamma, IL-10, and IL-2. CD4+CD25+ cell depletion increased IL-2 production by CD4+CD25- thymic cells, but not splenic cells. Moreover, tolerance was transferable with splenic and thymic CD4+CD25+ cells, but also in 50% of cases with splenic CD4+CD25- cells, demonstrating that CD25 can be a marker for regulatory cells in the thymus, but not in the periphery. In addition, we presented evidences that donor APCs were required to induce tolerance and to expand regulatory CD4+ T cells. This study demonstrates that LF15-0195 treatment induces donor APCs to expand powerful regulatory CD4+CD25+/- T cells present in both the central and peripheral compartments.

Addition of an IL-15 mutant/FCγ2A antagonist protein protects islet allografts from rejection overriding costimulation blockade

IL-15, a powerful chemoattractant and growth factor for antigen-activated T cells and NK cells, contributes to lymphocyte homeostasis and selectively induces the activation and proliferation of memory CD8+ T cells.1,2 Analysis of the phenotype of IL-15−/− mice3 and IL-15Rα−/− mice4 demonstrate that IL-15 is a critical factor for innate and adaptive immune responses. IL-15 is produced by the monocyte/macrophage lineage, by epithelial and endothelial cells but not by T-cells. The IL-15 receptor (IL-15R) expressed upon T cells is composed of three chains. Two are constitutively expressed and shared with the IL-2 receptor (IL-2R), the IL-2Rβ and IL-2Rγc chains, and a unique IL-15Rα chain is expressed upon activation of, but not during resting of mononuclear leukocytes.5,6 Because IL-15 and IL-2 share two receptor signaling components (IL-2/IL-15Rβγc), they display similar biological properties in vitro. Nevertheless, there are differential patterns of IL-15/IL-15Rα expression and in vivo studies have revealed major nonredundant roles for these two T-cell growth factors (TCGFs). In organ transplantation, T cells play a critical role in initiating the immune response leading ultimately to the effector mechanisms mediating allograft rejection. An analysis of biopsies taken from rejecting human renal allografts has revealed that intragraft gene expression of IL-15 is a far more consistent marker of rejection than IL-2 gene expression.7 Moreover, IL-2 expression is not a prerequisite for allograft rejection as IL-2 knockout (KO) mice promptly reject islet and cardiac allografts.7,8 Immunohistology in the IL-2 KO recipients reveals infiltration of rejecting grafts by CD4 and CD8 T cells as well as robust intragraft expression of the IL-4, IL-7, and IL-15 as well as granzyme B genes.8 Since costimulation blockade drastically decreases post-transplant expression of IL-2,9 the clonal proliferation of alloreactive T cells in hosts treated with costimulation blockade may be explained by the action of a non-T-cell-derived cytokine, such as IL-15. Although costimulatory blockade of CD28/CTLA4/B7 or CD40/CD40L interactions reduces immune activation and provides a powerful inhibition of alloimmune responses in both rodents and primates,9–11 in some circumstances it failed to induce permanent engraftment. Several reports suggest that CD8+ T cells are responsible for costimulation blockade-resistant rejection in various models, such as intestinal and skin allograft models.12,13 For example, adjunctive treatment with anti-CD8 mAb or other agents to deplete CD8+ T cells, are required to induce tolerance in these models.14 The resistance of CD8+ T cells to costimulation blockade may be explained by a poor effect of CTLA4Ig and αCD154 Abs to regulate activation and proliferation of CD8+ compared to CD4+ T cells.14,15 Recently, Smith et al16 have reported the prolonged survival of heart allografts in a treatment protocol consisting of soluble IL-15 receptor- α chain proteins and nondepleting anti-CD4 mAb. Thus the expression of IL-15 may be linked to CD8-dependent costimulation-resistant rejection. As costimulation blockade resistant rejection is mediated by CD8+ T cells and as IL-15 is essential for the activation and proliferation of CD8+ T cells, we hypothesized that targeting the IL-15/IL-15R system in conjunction with costimulation blockade would provide a new perspective for inducing allograft tolerance. The development of agents targeting the receptor and signaling elements of IL-15 may provide new perspectives for treatment of diseases associated with the IL-15/IL-15R pathway. The idea of creating an IL-15 antagonist was based on the homology of glutamine residues within the C-terminus of the four helix structures shared by IL-2 and IL-15, and on the report that a C-terminal glutamine in IL-2 is crucial for IL-2 binding to IL-2Rγ chain.17 In our laboratory a novel IL-15 mutant/Fcγ2a protein was genetically constructed, linked to murine Fcγ2a and expressed in mammalian cells. The IL-15 mutant was constructed by replacing the codons for the C-terminal glutamine amino acid residues with codons for aspartic acid (ie, Q101 and Q108).18 The mutant IL-15 moiety is a high-affinity IL-15R site-specific antagonist. The Fc portion of this fusion protein confers longevity and is designed to support cytocidal activities against IL-15R α+ cells insofar as the Fc-related sequences of mouse IgG2a isotype that support complement activation and activation of FcR+ phagocytes are present.18 Moreover, this IL-15 mutant/Fcγ2a protein competitively inhibits IL-15-triggered cell proliferation and does not activate the STAT-signaling pathway. Because the receptor site-specific antagonist IL-15 mutant/Fcγ2a protein had a prolonged half-life in vivo and the potential for destruction of IL-15R + leukocytes, the immunosuppressive effects of this agent were examined in mice with induced delayed-type hypersensitivity (DTH) and collagen-induced arthritis (CIA), a murine model for RA. In the DTH model, treatment with IL-15 mutant/Fcγ2a protein attenuated DTH responses and mononuclear leukocyte infiltration within the DTH sites.18 In the CIA model, treatment with IL-15 mutant/Fcγ2a protein markedly decreased the incidence and severity of arthritis in mice (S. Ferrari-Lacraz, inpreparation). As this unique IL-15 mutant/Fcγ2a protein displays promising effects on T-cell-derived pathogenesis, we wanted to determine its role and relative merits as a new therapeutic strategy in transplantation.

Induction of donor-specific tolerance to islet allografts in nonhuman primates.

Pancreatic tissue grafting is by far the most physiological therapeutic solution to the insulin deficiency of diabetes. Recent clinical trials have indicated somewhat successful use of nonsteroidal immunosuppressive regimens and a successful nonhuman primate trial using CD154 for costimulation blockade was reported. However, these protocols need to be replaced with safe and efficacious ones in which long-term allotolerance would make these treatments routine in a clinical setting. With the specific objective of testing whether peripheral infusions of stem cells or stem cell fractions in conjunction with islet allografting would induce allograft tolerance, we have established a macaque diabetic model. The macaques were rendered diabetic by streptozotocin and required daily doses of insulin to maintain lower blood glucose levels. The diabetic macaques then received islets and stem cells from unrelated and MHC-mismatched donors without any immunosuppression. In our initial analysis, 5 of 7 macaques that received stem cell infusions at the time of islet allografting have shown allograft survival longer than the group of macaques that received islets without the stem cell infusion. One of these five macaques has been normoglycemic for 10 months, with no exogenous insulin. This macaque received stem cell population enriched for CD34+ cells with depletion of CD18 cells, which have shown low or no allostimulatory potential in mixed lymphocyte cultures. Increased levels in insulin and C-peptide levels were shown in the macaques after islet transplantation.

Modern immunosuppressives

Eds HJ Schuurman , G Feutren & JF Bach . Published Birkhäuser Verlag AG 2001 . ISBN 3-7643-5981-1 This volume is a monograph in the series ‘Milestones in Drug Therapy’ and reviews present knowledge of modern immunosuppressive drugs. After an introductory chapter by Sir Roy Calne and David White, which reviews the history of immunosuppressive drugs, the book is divided into four sections: Immunosuppressive Drugs, Biologicals, New Avenues in Immunosuppression, and Perspectives. The chapter by Calne & White, who have collaborated over many years in the development and clinical application of new immunosuppressive drugs, nicely sets the scene for the remaining chapters. The section on immunosuppressive drugs covers cyclosporin, tacrolimus, sirolimus, and mycophenolate mofetil. The section on Biologicals covers the therapeutic use of monoclonal antibodies, and the targeting of IL-2 receptors with antibodies or chimeric toxins. New Avenues in Immunosuppression looks at emerging therapeutic strategies for the prevention and treatment of chronic allograft rejection, tolerance induction, and gene therapy approaches to immunosuppression. The final section on Perspectives looks at synergy among combinations of immunosuppressive therapies and the use of cyclosporin in the treatment of autoimmune diseases. The authors state in the preface that this monograph has been compiled for the use of clinicians working with immunosuppressive drugs in daily patient care and for researchers in preclinical and clinical studies. As a transplant surgeon, I found the chapters somewhat uneven, some being written with the clinician in mind and others very much for the research worker. On the whole, the chapter in the section on immunosuppressive drugs is well written indicating their pharmacological properties and their clinical benefits are clearly stated. However, the chapter dealing with tacrolimus ends with a short section comparing tacrolimus with cyclosporin, questioning which agent to use but does not satisfactorily answer this question and does not indeed fully discuss the incidence of side-effects of each drug in comparison with the other. The chapter on emerging therapeutic strategies for the prevention and treatment of chronic allograft rejection is well worth reading being crisply written and extremely well referenced. The chapter by Chou & Kahan on the approach to synergy in combination therapy contains complicated mathematics and is clearly written for the research worker although the last few pages of the chapter provide a summary of drug interactions which will be of value to clinicians. The final chapter is an overview on the use of cyclosporin in the treatment of autoimmune disease. This looks at the incidence of adverse effects when using cyclosporin in autoimmune disease, which apparently differ from those observed in clinical transplantation. I found this chapter somewhat out of place in this volume and I think it would also have benefited from some linguistic revision by the editors. Overall, this is a well-referenced and up-to-date volume and there is something in this book for everybody involved in the development and use of immunosuppressive agents. However, there are several other books available to clinicians on the subject of immunosuppressive agents and I suspect that this book will find its niche with the research worker rather than with the clinician.

New approaches to inducing the death of alloreactive lymphocytes

New approaches to inducing the death of alloreactive lymphocytes

Death of alloreactive T cells sets the stage for immunoregulation to act

WHILE MANY tolerizing treatment protocols use broadly reactive lymphocyte-depleting antibodies or achieve clone-specific depletion via the clonal depletive effect of the mixed chimeric state, nondepleting agents such as nondepleting anti-CD4 or anti-CD3mAb, costimulation blockade, rapamycin, or combined costimulation blockade plus rapamycin therapy can also induce tolerance. We have determined that induction of allograft tolerance achieved through use of nondepleting lymphocyte agents requires activation-induced cell death (AICD) and passive cell death (PCD) of alloreactive T-cell clones. If AICD, a process that is IL-2-dependent, is blocked by the potent immunosuppressive combination of calcineurin inhibitors plus costimulation blockade, a combination that blocks both signal 1 and 2 of T-cell activation, peripheral tolerance is not achieved. IL-2 KO mice that accumulate activated T cells in peripheral lymphoid tissues as a result of a failure to mount IL-2-dependent AICD are refractory to tolerance induction by rapamycin or by agents that block costimulation. If PCD is blocked via overexpression of antiapoptotic genes in T-cells, peripheral tolerance is not achieved. Thus, at least some nondepleting treatments require T-cell depletion via AICD and PCD for tolerance induction. A minimal requirement for tolerance is the depletion of alloaggressive T-cell (trim the pool size) clones. Regulatory T cells can readily be detected in many tolerized allograft recipients. In essence, we believe that the outcome of the allograft response, be it rejection or tolerance, depends upon the speed with which the pool of donor-specific alloaggressive or counteracting pool of T-reg cells expand posttransplantation. In the absence of effective treatment, the pool of alloaggressive clones expands more rapidly than the pool of T-reg cells; rejection is the result.

Dissociation of hemopoietic chimerism and allograft tolerance after allogeneic bone marrow transplantation.

Creation of stable hemopoietic chimerism has been considered to be a prerequisite for allograft tolerance after bone marrow transplantation (BMT). In this study, we demonstrated that allogeneic BMT with bone marrow cells (BMC) prepared from either knockout mice deficient in both CD4 and CD8 T cells or CD3E-transgenic mice lacking both T cells and NK cells maintained a high degree of chimerism, but failed to induce tolerance to donor-specific wild-type skin grafts. Lymphocytes from mice reconstituted with T cell-deficient BMC proliferated when they were injected into irradiated donor strain mice, whereas lymphocytes from mice reconstituted with wild-type BMC were unresponsive to donor alloantigens. Donor-specific allograft tolerance was restored when donor-type T cells were adoptively transferred to recipient mice given T cell-deficient BMC. These results show that donor T cell engraftment is required for induction of allograft tolerance, but not for creation of continuous hemopoietic chimerism after allogeneic BMT, and that a high degree of chimerism is not necessarily associated with specific allograft tolerance.

Allorecognition

Until recently, the vigorous T cell response via the direct pathway has overshadowed studies involving the indirect pathway. Thus, while the direct pathway has previously been considered to be the main driving force in alloimmune responses, there is an increasing body of data to support a prominent role of the indirect pathway in transplant rejection. Most importantly, while the direct anti-donor alloresponse diminishes with time after transplantation, the indirect alloresponse is likely to be permanently active due to traffic of recipient dendritic cells (DCs) through the graft. Consequently, the future challenge in the induction of allograft tolerance is to design interventions that will target the cells involved in the indirect pathway, especially the T cells with indirect allospecificity.

Il-12 antagonism enhances apoptotic death of T cells within hepatic allografts from Flt3 ligand-treated donors and promotes graft acceptance.

Mouse livers are accepted across MHC barriers and induce donor-specific tolerance without immunosuppressive therapy. By contrast, livers from donors treated with Flt3 ligand, which dramatically increases hepatic interstitial dendritic cells, are rejected acutely (median survival time 5 days). This switch from tolerance to rejection is associated with a marked reduction in apoptotic activity of graft-infiltrating cells. We hypothesized that IL-12 production by enhanced numbers of donor APC might inhibit apoptosis, promote expansion of Th1 cells, and play a key role in liver rejection. Therefore, C3H (H2(k)) recipients of liver grafts from Flt3 ligand-treated B10 donors were given neutralizing anti-IL-12 mAb (200 or 500 microg) on days 0 and 2 after transplant. Graft survival was markedly prolonged at the higher mAb dose, with 50% of grafts surviving >100 days. This effect was associated with reductions in IFN-gamma gene transcripts within the graft-infiltrating cell population and with reductions in circulating IFN-gamma and IL-10 levels, donor-specific CTL and NK cell activities, and circulating alloantibody levels. At the same time, there were marked increases in apoptotic (TUNEL(+)) CD4(+) and especially CD8(+) cells, both within the grafts and in spleens of anti-IL-12 mAb-treated mice. In vitro, exogenous IL-12 inhibited apoptotic death induced in naive allogeneic T cells by liver nonparenchymal cells. These findings suggest that suppression of rejection by IL-12 antagonism, linked to restoration of apoptotic activity within the peripheral alloreactive T cell population, is important for liver allograft survival and tolerance induction.