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Activation of innate immunity through Toll-like receptors (TLR) can abrogate transplantation tolerance by revealing hidden T cell alloreactivity. Separately, the cholinergic anti-inflammatory pathway has the capacity to dampen macrophage activation and cytokine release during endotoxemia and ischemia reperfusion injury. However, the relevance of the α7 nicotinic acetylcholine receptor (α7nAChR)-dependent anti-inflammatory pathway in the process of allograft rejection or maintenance of tolerance remains unknown. The aim of our study is to investigate whether the cholinergic pathway could impact T cell alloreactivity and transplant outcome in mice. For this purpose, we performed minor-mismatched skin allografts using donor/recipient combinations genetically deficient for the α7nAChR. Minor-mismatched skin grafts were not rejected unless the mice were housed in an environment with endogenous pathogen exposure or the graft was treated with direct application of imiquimod (a TLR7 ligand). The α7nAChR-deficient recipient mice showed accelerated rejection compared to wild type recipient mice under these conditions of TLR activation. The accelerated rejection was associated with enhanced IL-17 and IFN-γ production by alloreactive T cells. An α7nAChR-deficiency in the donor tissue facilitated allograft rejection but not in recipient mice. In addition, adoptive T cell transfer experiments in skin-grafted lymphopenic animals revealed a direct regulatory role for the α7nAChR on T cells. Taken together, our data demonstrate that the cholinergic pathway regulates alloreactivity and transplantation tolerance at multiple levels. One implication suggested by our work is that, in an organ transplant setting, deliberate α7nAChR stimulation of brain dead donors might be a valuable approach for preventing donor tissue inflammation prior to transplant.

Calcineurin-inhibitor refractory bronchiolitis obliterans (BO) represents the leading cause of late graft failure after lung transplantation. T helper (Th)2 and Th17 lymphocytes have been associated with BO development. Taking advantage of a fully allogeneic trachea transplantation model in mice, we addressed the pathogenicity of Th cells in obliterative airway disease (OAD) occurring in cyclosporine A (CsA)-treated recipients. We found that CsA prevented CD8(+) T cell infiltration into the graft and downregulated the Th1 response but affected neither Th2 nor Th17 responses in vivo. In secondary mixed lymphocyte cultures, CsA dramatically decreased donor-specific IFN-γ production, enhanced IL-17 production and did not affect IL-13. As CD4(+) depletion efficiently prevented OAD in CsA-treated recipients, we further explored the role of Th2 and Th17 immunity in vivo. Although IL-4 and IL-17 deficient untreated mice developed an OAD comparable to wild-type recipients, a single cytokine deficiency afforded significant protection in CsA-treated recipients. In conclusion, CsA treatment unbalances T helper alloreactivity and favors Th2 and Th17 as coexisting pathways mediating chronic rejection of heterotopic tracheal allografts., (© Copyright 2013 The American Society of Transplantation and the American Society of Transplant Surgeons.)

Background: Antibodies to platelet (PLT) glycoprotein (GP) IV (CD36) have been implicated in rare cases of PLT refractoriness, particularly in non-Caucasians. We report two cases of PLT transfusion refractoriness linked to anti-CD36., Study Design and Methods: A 5-year-old female of Lebanese descent and a 70-year-old male of Chinese descent both failed to respond to HLA-matched PLT transfusions during acute myelogenous leukemia induction therapy. Antibody screening was performed using a PLT antibody solid-phase kit (PAKPLUS, GTI Diagnostics), followed by the monoclonal antibody-specific immobilization of PLT antigen (MAIPA) test and, for the second case, the modified antigen capture enzyme-linked immunosorbent assay (MACE)., Results: Both patients demonstrated antibody to GP IV (CD36) on the PAKPLUS assay. On MAIPA testing, both phenotyped as CD36 negative. Anti-CD36 was demonstrated by MAIPA in the first case. In the second case, antibodies were not detected by MAIPA and variably detectable by MACE, depending on the mouse monoclonal antibody (MoAb) used. Because no Canadian CD36-negative donors were available, antigen-negative plateletpheresis units from the BloodCenter of Wisconsin were successfully transfused., Conclusion: Two cases of clinically significant CD36 antibodies are reported. Investigation of one case was complicated by steric inhibition of binding in the MAIPA and MACE assays with certain MoAbs. The cases demonstrate the importance of maintaining an ethnically diverse pool of rare donors and the value of international cooperation in the management of these patients., (© 2010 American Association of Blood Banks.)

The long-term stability of renal grafts depends on the absence of chronic rejection. As T cells play a key role in rejection processes, analyzing the T-cell repertoire may be useful for understanding graft function outcomes. We have therefore investigated the power of a new statistical tool, used to analyze the peripheral blood TCR repertoire, for determining immunological differences in a group of 229 stable renal transplant patients undergoing immunosuppression. Despite selecting the patients according to stringent criteria, the patients displayed heterogeneous T-cell repertoire usage, ranging from unbiased to highly selected TCR repertoires; a skewed TCR repertoire correlating with an increase in the CD8(+) /CD4(+) T-cell ratio. T-cell repertoire patterns were compared in patients with clinically opposing outcomes i.e. stable drug-free operationally tolerant recipients and patients with the "suspicious" form of humoral chronic rejection and were found significantly different, from polyclonal to highly selected TCR repertoires, respectively. Moreover, a selected TCR repertoire was found to positively correlate with the Banff score grade. Collectively, these data suggest that TCR repertoire categorization might be included in the calculation of a composite score for the follow-up of patients after kidney transplantation., (Copyright © 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Th17-mediated immune responses have been recently identified as novel pathogenic mechanisms in a variety of conditions; however, their importance in allograft rejection processes is still debated. In this paper, we searched for MHC or minor Ag disparate models of skin graft rejection in which Th17 immune responses might be involved. We found that T cell-derived IL-17 is critical for spontaneous rejection of minor but not major Ag-mismatched skin grafts. IL-17 neutralization was associated with a lack of neutrophil infiltration and neutrophil depletion delayed rejection, suggesting neutrophils as an effector mechanism downstream of Th17 cells. Regulatory T cells (Tregs) appeared to be involved in Th17 reactivity. We found that in vivo Treg depletion prevented IL-17 production by recipient T cells. An adoptive cotransfer of Tregs with naive monospecific antidonor T cells in lymphopenic hosts biased the immune response toward Th17. Finally, we observed that IL-6 was central for balancing Tregs and Th17 cells as demonstrated by the prevention of Th17 differentiation, the enhanced Treg/Th17 ratio, and a net impact of rejection blockade in the absence of IL-6. In conclusion, the ability of Tregs to promote the Th17/neutrophil-mediated pathway of rejection that we have described should be considered as a potential drawback of Treg-based cell therapy.

We designed a pilot trial in cadaveric liver transplantation to determine whether induction with antithymocyte globulins (ATG) and sirolimus would allow immunosuppression withdrawal. Patients received ATG 3.75 mg/kg per day from day 1 to 5 after transplantation followed by sirolimus for 4 to 6 months. We monitored interleukin (IL)-7 serum levels, interferon (IFN)-gamma, and IL-2 mRNA accumulation in mixed leukocyte reaction and intragraft IFN-gamma mRNA expression. In the first three patients, immunosuppression discontinuation was followed by reversible acute rejection occurring on days 280, 246, and 163 posttransplantation, corresponding to days 140, 40, and 39 after drug withdrawal, respectively. At the time of rejection, blood CD8+ T-cells counts had returned to or above pretransplant levels in two of three patients, whereas CD4+ T-cell count remained low. IL-7 serum levels rose in all three patients in the first months after transplantation and IFN-gamma mRNA accumulated in mixed leukocyte reaction between recipient T cells and donor spleen cells at the time of rejection. High levels of IFN-gamma mRNA were consistently detected in liver biopsy performed at the time of rejection. In conclusion, lymphopenia-induced IL-7 production after induction with ATG and sirolimus might lead to emergence of IFN-gamma-secreting CD8+ T-cells responsible for acute rejection after immunosuppression withdrawal.

Interleukin-22 (IL-22) was recently described as an effector cytokine produced by TH17 CD4(+) T lymphocytes that, cooperatively with IL-17, mediates IL-23-driven inflammation. Because there was experimental evidence for the role of IL-17 in acute rejection of vascularized allografts, we undertook the present study to assess the function of IL-22 in the process. There was an early transient expression of IL-22 in C57BL/6 mouse cardiac allografts (2-4 days posttransplantation) transplanted to BALB/c recipients. The main source of IL-22 among infiltrating leukocytes was cells expressing the macrophage/monocyte markers Mac3 and CD11b. T cells and granulocytes present in the rejected graft did not express IL-22. Surprisingly, the absence of IL-22 accelerated the rejection of fully histoincompatible hearts. Histology of rejected organs revealed the presence of intensive intragraft thrombosis and disseminated hemorrhagic necrosis. Taken together, these results demonstrated that IL-22 was not an effector lymphokine in cardiac allograft rejection, but early intragraft expression of the cytokine protected it from rejection.

Purpose of Review: Allograft rejection involves multiple effector mechanisms. Interleukin(IL)-12 family members play a critical role in influencing helper T-cell differentiation and inflammatory processes, and their respective role in orchestrating inflammation of autoimmune or infectious origin starts to be unravelled. We highlight recent findings on the function of the different IL-12 family members: IL-12p70, IL-23, IL-27 and IL-35 and discuss their possible involvement in influencing the balance between graft rejection and tolerance., Recent Findings: The capacity of dendritic cells to produce IL-12 and IL-23 strongly influences the outcome of CD4 T-cell responses. While the IL-12/interferon-gamma axis has classically been involved in autoimmune pathologies and acute graft rejection, it is now clear that it also displays immunoregulatory properties. In contrast, IL-23 promotes the function of proinflammatory IL-17-producing cells in both mice and humans. Both IL-27 and IL-35 have recently emerged as important regulators of adaptive immune responses., Summary: The contribution of the IL-12/interferon-gamma pathway to acute graft rejection may be more complicated than initially thought. As our understanding of the IL-12 family is rapidly growing and changing, the respective role of its members in orchestrating innate and adaptive immune responses toward alloantigens should be addressed.

Rejection of transplanted organs depends on an orchestrated immune response to histocompatibility antigens expressed by the grafted tissue. Effector mechanisms primarily responsible for the rejection process classically involve type 1 helper CD4(+) T cells, cytotoxic CD8(+) T cells and antibodies. Experimental studies revealed alternative mechanisms of rejection that implicate type 2 helper CD4(+) T cells and memory CD8(+) T cells as well as cells belonging to the innate immune system including natural killer cells, eosinophils and neutrophils. Furthermore, local inflammation associated with rejection is tightly regulated at the graft level by regulatory T cells and mast cells. The redundancy of rejection mechanisms explains the difficulty to induce transplantation tolerance and to develop reliable biomarkers for prediction of allograft outcome.

Since several years ago, interleukin (IL)-12 is known to be responsible for the differentiation of naive CD4+ T cells into type 1 helper T cells producing interferon-gamma. Recently, two other cytokines of the IL-12 family, IL-23 and IL-27, were shown to play key roles in experimental autoimmune disorders mediated by Th17 cells, a novel pro-inflammatory CD4+ T-cell subset secreting IL-17. As our knowledge of IL-12 family members is rapidly growing and changing, it will be important to specify their involvement in the induction and regulation of allograft rejection in animal models as well as in clinical settings. Herein, we review key features of cytokines belonging to the IL-12 family and discuss their potential relevance to transplantation immunity.

Background: Whereas the participation of alloreactive T cells sensitized by indirect allorecognition in graft rejection is well documented, the nature of recipient antigen presenting cells recognized by indirect pathway CD4+ T cells within the graft has yet to be identified. The purpose of this study was to determine the role played by graft endothelium replacement in the immune recognition of cardiac allografts rejected by indirect pathway CD4+ T cells., Methods: Transgenic RAG2-/- mice expressing I-Ab-restricted male antigen H-Y-specific TcR were studied for their capacity to reject H-2k male cardiac allografts. Chronic vascular rejection in this model was due to the indirect recognition of H-Y antigen shed from H-2k male allograft and presented by the recipient's own I-Ab APC to transgenic T cells., Results: Immunohistochemical analysis of rejected grafts revealed the presence of numerous microvascular endothelial cells (EC) that expressed recipient's I-Ab MHC class II molecules. This observation suggested that graft endothelium replacement by I-Ab-positive cells of recipient origin could stimulate the rejection of male H-2k graft by I-Ab-restricted H-Y-specific T cells. To investigate further this possibility, hearts from H-2b-into-H-2k irradiation bone marrow (BM) chimera were transplanted in transgenic recipients. A direct correlation was observed between the presence of I-Ab-positive EC within myocardial microvessels and the induction of acute rejection of chimeric H-2k male cardiac allografts transplanted in transgenic recipients., Conclusions: We conclude that graft endothelium replacement by recipient-type cells is required for the rejection of cardiac allograft mediated by indirect pathway alloreactive CD4+ T cells.

Background: Acute rejection of MHC class II-disparate bm12 skin grafts by C57BL/6 recipient mice is characterized by massive graft infiltration by eosinophils, together with increased intragraft amounts of IL-4 and IL-5 mRNA. IL-5 blockade prevents the intragraft eosinophil infiltration and prolongs the survival of skin allografts. As the differentiation of T cell precursors into Th2 cells is largely driven by IL-4, we investigated the role of IL-4 in MHC class II-disparate allograft rejection., Methods: We performed skin grafts from MHC class II incompatible bm12 mice into wild-type C57BL/6 mice (IL-4) or C57BL/6 IL-4 deficient mice (IL-4). Graft survival, in vitro T cell reactivity, and histology were compared., Results: We observed that 50% of IL-4 mice rapidly rejected their bm12 allograft, whereas the other 50% retained their graft 60 days after transplantation. Histological examination of bm12 allografts retained by IL-4 mice showed a normal appearance with no inflammatory infiltrate and no eosinophils. Among IL-4 mice that acutely rejected their bm12 skin graft, we observed a dense polymorphonuclear infiltrate. The depletion of neutrophils significantly prolonged bm12 graft survival., Conclusions: Eosinophil infiltrates, typical of MHC class II disparate acute skin graft rejection, are critically dependent on the availability of IL-4. IL-4 mice reject MHC class II disparate skin grafts by a pathway of rejection where neutrophils play a direct causal role.

We recently showed that interleukin-9 (IL-9), a Th2 cytokine, promotes IL-5-mediated rejection of allografts in mice. This observation led us to investigate the functional link between IL-9 and IL-5 production during alloreactive T cell responses in vitro and in vivo. Firstly, we found that IL-9 was produced by alloreactive Th2 cells, and IL-9 mRNA was detected in skin allograft during Th2-type rejection. We then established that IL-5 production was impaired in alloreactive Th2 cells isolated from IL-9-deficient mice and that optimal IL-5 production after allogeneic stimulation requires a functional IL-9 receptor (IL-9R) on the responding cells. Finally, the production of IL-5 by anti-CD3-stimulated CD4+ T cells was abolished by neutralization of IL-9. Despite the fact that IL-9 promotes IL-5 production by alloreactive T cells, IL-9-deficient recipients of skin allografts still developed eosinophilic graft infiltrates and neither IL-9 nor IL-9R deficiency modified Th2-type allograft rejection.

Because rejection of allografts is primarily caused by T and B lymphocyte responses to allogeneic histocompatibility molecules, the role of innate immunity in organ transplant rejection is often overlooked. However, the very first damages to vascularized organ allografts are caused by ischemia-reperfusion, an inflammatory reaction involving activation of vascular endothelial cells and release of neutrophil chemoattractants. Herein, we review experimental observations suggesting that the early neutrophil influx in organ transplants favors T cell-mediated rejection.

Because rejection of allografts is primarily caused by T and B lymphocyte responses to allogenic histocompatibility molecules, the role of innate immunity in organ transplant rejection is often overlooked. However, the very first damages to vascularized organ allografts are caused by ischemia-reperfusion, an inflammatory reaction involving activation of vascular endothelial cells and release of neutrophil chemoattractants. Herein, we review experimental observations suggesting that the early neutrophil influx in organ transplants favors T cell-mediated rejection.

Background: Eosinophils participate in allograft rejection when donor-reactive helper T lymphocytes are T-helper type 2 (Th2)-biased. Whereas the involvement of interleukin (IL)-4 and IL-5 in these forms of rejection is well established, the role of IL-9, another Th2-type cytokine promoting eosinophilia, has not been determined., Methods: We first used real-time polymerase chain reaction to quantify IL-9 mRNA in rejected allografts in a mouse model of fully mismatched heart transplantation in which recipients were devoid of CD8 T cells and developed a Th2 alloimmune response. We then compared allograft survival in wild-type versus IL-9-deficient mice depleted of CD8 T cells. Finally, we compared the fate of major histocompatibility complex class II-mismatched cardiac transplants from wild-type versus IL-9 transgenic donors to determine the influence of IL-9 overexpression within the graft., Results: The Th2 alloimmune response in CD8-deficient mice was associated with the accumulation of IL-9 mRNA in the rejected graft. In IL-9-deficient recipients depleted of CD8 T cells, eosinophil infiltration of heart allografts did not develop, but rejection still occurred. In the major histocompatibility complex class II disparate model, heart allografts from IL-9 transgenic donors were acutely rejected, whereas grafts from wild-type donors did not develop rejection. Acute rejection of IL-9 transgenic hearts was associated with massive eosinophil infiltration and prevented by neutralization of either IL-4 or IL-5., Conclusion: IL-9 is critically involved in heart transplant eosinophilia in conjunction with IL-4 and IL-5.

Background: Dendritic cells (DC) at the immature state express low levels of major histocompatibility complex and costimulatory molecules and are poor stimulators of primary T-cell response in vitro. Injection of immature bone marrow-derived DC, however, was shown to prime in vivo alloreactive CD4 T lymphocytes toward type 2 cytokine-producing cells in the absence of CD8 T-cell activation., Methods: We undertook the present study to determine whether Th2-immunization by immature DC could lead to allograft rejection. We first analyzed, in the major histocompatibility complex class II antigen-disparate B6-anti-bm12 combination, the capacity of immature DC to regulate the activity of alloreactive CD4 T cells. We then determined, in this model of weak antigenicity, whether injection of bm12 DC in B6 recipients before transplantation could modify the survival of vascularized bm12 cardiac allografts., Results: We confirmed that in vitro immature DC are poor stimulators of T-cell alloresponse. However, when given in vivo, immature bm12 DC primed anti-bm12 T cells for the production of interleukin (IL)-4. Moreover, they induced the acute rejection of bm12 cardiac allograft. The process of rejection was dependent on IL-4 because immunization of IL-4-deficient mice did not trigger rejection., Conclusions: Allogeneic immature DC generated with granulocyte-macrophage colony-stimulating factor are potent stimulators of primary alloreactive response in vivo and prime for transplant rejection. Our results indicate that strategies based on immature DC for the induction of transplantation tolerance should be considered with caution.

Allograft rejection results from separate pathways primarily controlled by CD4+ T cells. Refinement of transplantation models together with investigations on rejection occurring despite co-stimulation blockade revealed unexpected pathways involving CD8+ T cells, NK cells and Th2 cytokines. In this minireview, we discuss these non-classical pathways of allograft rejection and their relevance for the induction of tolerance in the clinics.

Beta(2)-microglobulin (beta(2)m)-derived peptides are minor transplantation Ags in mice as beta(2)m-positive skin grafts (beta(2)m(+/+)) are rejected by genetically beta(2)m-deficient recipient mice (beta(2)m(-/-)). We studied the effector pathways responsible for the rejection induced by beta(2)-microglobulin-derived minor transplantation Ags. The rejection of beta(2)m(+/+) skin grafts by naive beta(2)m(-/-) mice was dependent on both CD4 and CD8 T cells as shown by administration of depleting mAbs. Experiments performed with beta(2)m(-/-)CD8(-/-) double knockout mice grafted with a beta(2)m(+/+) MHC class I-deficient skin showed that sensitized CD4 T cells directed at beta(2)m peptides-MHC class II complexes are sufficient to trigger rapid rejection. Rejection of beta(2)m(+/+) grafts was associated with the production of IL-5 in vitro, the expression of IL-4 and IL-5 mRNAs in the grafted tissue, and the presence within rejected grafts of a considerable eosinophil infiltrate. Blocking IL-4 and IL-5 in vivo and depleting eosinophils with an anti-CCR3 mAb prevented graft eosinophil infiltration and prolonged beta(2)m(+/+) skin graft survival. Lymphocytes from rejecting beta(2)m(-/-) mice also displayed an increased production of IFN-gamma after culture with beta(2)m(+/+) minor alloantigens. In vivo neutralization of IFN-gamma inhibited skin graft rejection. Finally, beta(2)m(+/+) skin grafts harvested from B6(lpr/lpr) donor mice, which lack a functional Fas molecule, survived longer than wild-type beta(2)m(+/+) skin grafts, showing that Fas-Fas ligand interactions are involved in the rejection process. We conclude that IL-4- and IL-5-dependent eosinophilic rejection, IFN-gamma-dependent mechanisms, and Fas-Fas ligand interactions are effector pathways in the acute rejection of minor transplantation Ags.

Allograft rejection results from a complex process involving both the innate and acquired immune systems. The innate immune system predominates in the early phase of the allogeneic response, during which chemokines and cell adhesion play essential roles, not only for leukocyte migration into the graft but also for facilitating dendritic and T-cell trafficking between lymph nodes and the transplant. This results in a specific and acquired alloimmune response mediated by T cells. Subsequently, T cells and cells from innate immune system function synergistically to reject the allograft through nonexclusive pathways, including contact-dependent T cell cytotoxicity, granulocyte activation by either Th1 or Th2 derived cytokines, NK cell activation, alloantibody production, and complement activation. Blockade of individual pathways generally does not prevent allograft rejection, and long-term allograft survival is achieved only after simultaneous blockade of several of them. In this review, we explore each of these pathways and discuss the experimental evidence highlighting their roles in allograft rejection.

We recently demonstrated that interleukin-5 and eosinophils mediate rejection of skin allografts when CD8+ T cell-dependent and Th1-type CD4+ T cell-dependent pathways are not functional. The purpose of this study was to determine whether a similar mechanism might be operative during rejection of rat islet xenografts in mice. First, we observed that eosinophils indeed infiltrate rejected islet grafts together with CD4+ and CD8+ T cells. CD8+ T cell depletion significantly enhanced graft survival and a further prolongation of islet function was obtained in combination with interferon-gamma neutralization. However, islet rejection characterized by prominent eosinophil and macrophage infiltration still occurred in this setting. Although eosinophil infiltrates were dramatically reduced in interleukin-5 deficient mice, the ability of these animals to reject islet xenografts under CD8+ T cell depletion and interferon-gamma neutralization was similar to that of wild-type mice. We conclude that in absence of CD8+ T cells and interferon-gamma, macrophages, but not eosinophils, mediate rejection of rat-to-mouse islet xenografts.

Eosinophils release inflammatory mediators and cationic proteins that are instrumental in the pathogenesis of allergic diseases such as bronchial asthma. Here, we review experimental observations indicating that eosinophils are also involved in the rejection of allografts. We propose that their role as effectors of transplant damage becomes crucial when classical pathways of rejection are inhibited and T helper 2 (Th2) cells dominate the alloimmune response.

We studied the effects of the indirect pathway of allograft recognition using T cells from TCR transgenic Marilyn mice, which recognize the male Ag H-Y in an I-A(b)-restricted fashion. The T cells are not alloreactive to the H-2(k) haplotype, because they are not activated when adoptively transferred into recombinase-activating gene-2(-/-) common gamma-chain(-/-) double-mutant H-2(k) male or female mice. However, skin from H-2(k) males, but not from H-2(k) females, is acutely rejected by recombinase-activating gene-2(-/-) transgenic female recipients. In vitro, Marylin spleen cells primed by H-2(k) skin grafting proliferated and secreted both IL-4 and IFN-gamma in response to H-2(k) male stimulators. However, the removal of H-2(b) APC from the responding population abolished the response. Taken together, these results show that the indirect recognition that triggers rejection in this model is due to the recognition of H-Y Ag shed from H-2(k) male allograft and presented by the recipient's own I-A(b) APC to transgenic T cells. This study demonstrates unequivocally the capacity of naive CD4(+) T cells to promote the rejection of allografts through mechanisms that involve indirect destruction of grafted tissues.

CD8(+) T lymphocytes are known to inhibit the development of eosinophilia and IL-5 synthesis in models of experimental lung disease. In transplantation, the rejection of fully mismatched cardiac allografts by recipients depleted of CD8(+) T cells is characterized by the recruitment of eosinophils in the rejected organs. We show here that this intragraft eosinophilia is dependent on the production of IL-5 since hearts transplanted into IL-5-deficient recipients depleted of CD8(+) cells did not contain eosinophils. More importantly, allograft survival was significantly extended in these animals. In mixed lymphocyte cultures (MLC), the presence of CD8(+) T cells in the responding cell population inhibited the secretion of IL-5. This inhibition was IFN-gamma dependent since adding neutralizing anti-IFN-gamma antibodies induced the production of IL-5. Furthermore, spleen cells isolated from IFN-gamma receptor (IFN-gammaR)-deficient mice secreted IL-5 upon allogeneic stimulation in primary MLC. In vivo, eosinophilia was observed in allografts rejected by IFN-gammaR-deficient recipients. On the contrary, grafts rejected by IFN-gammaR-deficient mice treated with neutralizing anti-IL-5 antibodies did not exhibit eosinophilic infiltration. Our study reveals the capacity of IL-5-secreting CD4(+) T cells and eosinophils to promote the rejection of heart allograft and demonstrates the importance of CD8(+) T cells and IFN-gamma in regulating this pathway of rejection.

CD4 T cells play a crucial role in the acute rejection of MHC class II-disparate skin allografts, mainly by Fas/Fas ligand-mediated cytotoxicity. Because recent observations indicate that eosinophils may be found within allografts rejected by CD4 T cells, we evaluated the role played by IL-5, the main eosinophil growth factor, and by eosinophils in the rejection of MHC class II-disparate skin grafts. C57BL/6 mice rapidly rejected MHC class II-disparate bm12 skin grafts. Rejected skins contained a dense, aggressive eosinophil infiltrate. Lymphocytes isolated from lymph nodes draining rejected bm12 skin were primed for IL-5 secretion, and IL-5 mRNA was present within rejected grafts. The IL-5/eosinophil pathway played an effector role in allograft destruction, because the rejection of bm12 skin was significantly delayed in IL-5-deficient mice as compared with wild-type animals. The role of the IL-5/eosinophil pathway was further investigated in MHC class II-disparate donor-recipient strains unable to establish Fas/Fas ligand interactions. Fas ligand-deficient gld/gld mice rejected bm12 skins, and bm12 mice rejected Fas-deficient lpr/lpr C57BL/6 skins. Neutralization of IL-5 prevented acute rejection in both combinations. We conclude that MHC class II-disparate skin allografts trigger an IL-5-dependent infiltration of eosinophils that is sufficient to result in acute graft destruction.

C57BL/6 mice injected with the 145-2C11 anti-CD3 mAb and grafted with MHC class II disparate bm12 skin develop a chronic rejection characterized by interstitial dermal fibrosis, a marked eosinophil infiltrate, and an obliterative intimal vasculopathy. Because these changes occur in the absence of alloreactive antibodies, we examined the contribution of cytokines in their pathogenesis. Chronically rejected grafts showed a marked accumulation of both IL-4 and IL-5 mRNA. Mixed lymphocyte reaction experiments established that mice undergoing chronic rejection were primed for IL-4, IL-5, and IL-10 secretion. In vivo administration of anti-IL-4 mAb completely prevented allograft vasculopathy as well as graft eosinophil infiltration and dermal fibrosis. Injection of anti-IL-5 mAb or the use of IL-5-deficient mice as recipients also resulted in the lack of eosinophil infiltration or dermal fibrosis, but these mice did develop allograft vasculopathy. Administration of anti-IL-10 mAb did not influence any histologic parameter of chronic rejection. Thus, in this model, IL-4- and IL-5-mediated tissue allograft eosinophil infiltration is associated with interstitial fibrosis. IL-4, but not eosinophils, is also required for the development of obliterative graft arteriolopathy.

To assess the effects of azathioprine withdrawal, renal recipients with grafts > 2 yr function (103 study patients) were tapered off azathioprine over a 1-yr period and compared to 69 patients 2 yr after transplant who were not tapered (controls). Of the 103 study patients, 16 (15%) were living-donor transplants and 87 were cadaveric. Of the 69 control patients, 9 (13%) were living related transplants and 60 were cadaveric. The mean HLA match for those tapered was 3.3 Ag and 3.1 Ag for those remaining on azathioprine. Two study patients restarted azathioprine on their own. Age, sex, and cause of renal failure in both groups was similar. Of the 101 study patients remaining, 9 (8.8%) returned to dialysis due to biopsy proven chronic rejection. There were no acute rejection episodes. Six of the 69 control patients (8.7%) also returned to dialysis for the same reason. Of the 92 patients who have completed the taper, 85 have been off azathioprine for six or more months. There was not a significant difference between the mean 12- and 24-month creatinine levels of the study patients (1.6 mg%, 1.7 mg%) and those of the controls (1.5 mg%, 1.8 mg%). The mean 12- and 24-month hematocritis of patients tapered (41.3%, 40.8%) were comparable with patients not tapered (42.3%, 42.8%). Of interest, the mean hematocritis of both study and control patients rose from 28.9% and 33.5%, respectively, to 41.3% and 42.3% 1 yr following entry into the study. The mean 12- and 24-month white blood counts of those tapered (8.9, 8.7) did not differ significantly from those continued on azathioprine (8.8, 8.8). In stable renal transplant patients on triple drug immunosuppression for at least 2 yr, azathioprine can be discontinued, in a tapered protocol, without an increased risk of graft loss or compromise of renal function.

The first injection of OKT3 in kidney transplant recipients activates the common pathway of coagulation. This may result in early thrombosis of graft vessels. To this day, the cells involved in this phenomenon have not been identified. The aim of this study was to investigate whether circulating monocytes participated in this OKT3-induced coagulopathy. The procoagulant activity (PCA) of circulating monocytes rose from (mean +/- SEM) 0.15 +/- 0.02 mU/mL to 0.40 +/- 0.05 mU/mL at 3 hours (P = .002) and 0.56 +/- 0.21 at 5 hours (P = .045) after the initial OKT3 injection. These monocytes displayed increased tissue factor expression at the same moments (mean flourescence intensity: 14 +/- 2 before OKT3 injection versus 54 +/- 14 at 3 hours, P = .008 and 34 +/- 7 at 5 hours, P = .01). Tissue factor mRNA was detected in blood by reverse transcriptase-polymerase chain reaction as early as 2 hours after OKT3 administration. The circulating monocytes also displayed a steady increase in membrane expression upregulation of ICAM-1, CD29, CD11b, and CD11c. In vitro experiments showed that OKT3 as well as 2 mitogenic, humanized anti-CD3 antibodies potently induced monocytic PCA whereas the 4 nonmitogenic anti-CD3 antibodies tested were over 1,000-fold less potent than OKT3. We conclude that (1) OKT3 induces in vivo tissue factor gene upregulation and membrane expression resulting in increased PCA of circulating monocytes; and (2) nonmitogenic anti-CD3 antibodies seem devoid of significant procoagulant properties.

The data on patients participating in two randomized, prospective studies with similar immunosuppressive regimens were updated and combined to evaluate the long-term effects of OKT3 according to cold ischemia time (< or = or > 24 hr). Among 159 patients in the OKT3 and 153 in the cyclosporine A (CsA) group, 8 and 12 deaths occurred, respectively (P = NS). In patients with cold ischemia > 24 hours, OKT3 prophylaxis resulted in a lower mean number of rejection episodes per patient than did CsA prophylaxis within one year (mean +/- SEM: 0.87 +/- 0.11 vs. 1.35 +/- 0.14, respectively; P = 0.008) and within five years (1.07 +/- 0.12 vs. 1.49 +/- 0.15, respectively; P = 0.032). In contrast, rejection incidences in patients with cold ischemia < or = 24 hours was not significantly different in the two groups. In all study patients, there was a trend towards higher graft survival rates in the OKT3 group versus the CsA group (at 5 years, 73% vs. 66%, respectively; P = 0.182). Among recipients of kidneys with cold ischemia times > 24 hours, OKT3 patients had significantly higher graft survival than CsA patients at two years (84% vs. 64%, respectively) and at five years (71% vs. 56%, respectively; P = 0.045). Significant differences were not observed in recipients of kidneys with cold ischemia times < or = 24 hours. In conclusion, patients receiving renal grafts with long cold ischemia times strongly benefit from OKT3 prophylaxis.

In our experience the use of OKT3 as prophylaxis in renal transplantation has been associated with an increased incidence of both delayed graft function and thromboses of graft vessels. OKT3 nephrotoxicity might have been favored by restriction of perioperative fluid infusion to prevent pulmonary edema and by the use of very high dose (30 mg/kg) of methylprednisolone (mPDS) before the first OKT3 injection to reduce the release of cytokines. This led us to modify our perioperative management in three ways: (1) hydration status was optimalized; (2) the calcium-channel blocker diltiazem, considered beneficial for recovery of graft function, was administered on the day of transplantation; and (3) the dose of mPDS given before the first OKT3 injection was fixed at 8 mg/kg. Comparison of two consecutive series of patients (group 1, control patients, N = 172; group 2, managed as described above, N = 173) showed that: (1) the incidence of delayed graft function fell from 52% in group 1 to 22% in group 2 (P < 0.0001): (2) the incidence of pulmonary edema was not significantly increased in group 2 (3.5% vs. 1.7% in group 1, P = 0.5); and (3) the frequency of intragraft thrombosis fell from 7.6% in group 1 to 1.2% in group 2 (P = 0.0034). Multivariate analysis showed that the volemia/diltiazem program and avoidance of high mPDS dose were the most important factors responsible for the reduced occurrence of delayed graft function and graft vessels thrombosis, respectively. We conclude that a combined strategy of appropriate dosage of steroids before the first OKT3 injection, administration of a calcium-channel blocker and optimalization of volemia is safe and efficiently prevents against OKT3 nephrotoxic effects.

Previous studies from our center have shown that donor-recipient HLA-DR mismatches (MM), characterized by the presence of the DR antigen in the donor but not in the recipient or vice versa, are associated with differential effects on graft survival (GS): some of them are beneficial (BEN) with results similar to those of HLA-DR identical or compatible pairs (85% 18 months GS) and some are detrimental (DET) (64% 18 months GS), whereas the other MM, neither BEN nor DET (neutral [NEU]) yield intermediate results (78% 18 months GS). The aim of the present study was to update the results at a longer follow-up time and to assess whether they are influenced or not by prophylactic administration of anti-CD3 mAb (OKT3). The analysis of 234 transplantations performed from 1980 to 1994 with only 1 HLA-DR MM confirmed the BEN effects of HLA-DR5 in either the donor or the recipient and the DET effects of HLA-DR1 or -DR2 in the donor and of HLA-DR2 or -DRW6 in the recipient. These effects were independent of those exerted by other, HLA-DR not related, prognostic factors. The transplants with 1 HLA-DR MM were then compared with those with zero HLA-DR MM (n = 378) and 4 groups were formed according to 2 levels of HLA-DR MM (zero or BEN MM vs. NEU or DET MM) and immunosuppression (with vs. without OKT3 prophylaxis). GS at 5 years was 63% in the group with either zero or BEN MM as compared with 41% in the group with either NEU or DET MM in the absence of OKT3 prophylaxis (P < 0.02); in comparison, with OKT3 prophylaxis, GS at 5 years was 73% in the group with either zero or BEN MM as compared with 58% in the group with either NEU or DET MM (P = 0.07). We conclude that the differential effects of HLA-DR MM on GS are still observed under OKT3 prophylaxis, that the effects of HLA-DR and immunosuppression on graft outcome are additive, and that OKT3 induction therapy is superior to therapy without OKT3. These observations could have important implications for the allocation policy and management of renal transplants.

The use of OKT3 as prophylaxis in renal transplantation results in a reduced incidence of graft rejection and appears to have beneficial effects on long-term kidney graft survival. However, we and others have observed that patients still experience rejection during the period of OKT3 prophylaxis given at the regular 5 mg/day dose. Many of these patients had no circulating CD3+ cells at the time of rejection, but their OKT3 serum levels were distinctly low (< 500 ng/ml). This led us to adjust OKT3 doses (5 or 10 mg) daily, according to the patients' OKT3 levels, in order to maintain an OKT3 concentration of around 1000 ng/ml. In addition, patients were randomized to receive either 5 mg (group 1, n = 15) or 10 ng (group 2, n = 14) OKT3 as the initial three doses. Concomitant immunosuppression consisted of azathioprine and steroids, with the introduction of cyclosporin A on day 11. Patient survival was 100% after 3 months of follow-up. The intensity of OKT3 first-dose reactions was similar in both groups. Intragraft thrombosis, initially observed in a previous group of patients who received a fixed 10 mg/day OKT3 prophylaxis, occurred in three patients in group 1 and resulted in two graft losses. The cumulative OKT3 dose was similar in both groups (mean +/- SEM 98 +/- 2 mg in group 1 vs 102 +/- 3 mg in group 2) and higher than the 70 mg usually administered. Group 2 patients had higher OKT3 serum levels during the first 4 days of therapy. No correlation could be found between patient weight and cumulative OKT3 dose (r = 0.29).(ABSTRACT TRUNCATED AT 250 WORDS)

In this prospective randomized study, acute renal transplant rejections occurring in patients who received prophylactic OKT3 therapy were treated with either 3 pulses of 8 mg/kg methylprednisolone (MPS) in an alternate-day regimen (total dose 25 mg/kg in 1 week, H group, n = 24) or 5 daily pulses of 3 mg/kg MPS (total dose 17 mg/kg, L group, n = 22). Acute rejection was proven by biopsy in more than 85% of cases in both groups. No difference was observed in rejection reversal (H 88%, L 91%), graft losses in the following 3 months (H 11%, L 4%) or the time evolution of the serum creatinine levels. The number (H 14, L 21) as well as the nature and severity of infections were similar in both groups. Only one death occurred in a patient who received OKT3 rescue therapy for corticoresistant rejections and developed Epstein-Barr virus (EBV)-related lymphoma. In conclusion, low dose MPS pulses appear as effective and safe as a higher dose to reverse acute rejection occurring after OKT3 prophylaxis. Thus, we favour the use of the low dose regimen in these patients.

A 25-year-old recipient of a cadaveric renal allograft underwent three acute rejection episodes within the first 40 days after transplant, the final episode necessitating nephrectomy. The saralasin acetate infusion test and plasma renin activity (as measured by radioimmunoassay of angiotensin I) were used as functional tests of the renin-angiotensin axis. Biopsy specimens of the allograft one hour after implantation and sections of the nephrectomy specimen were fixed and stained with hematoxylin-eosin for structural analysis. On three separate dates, during the final rejection episode, saralasin acetate infusion of up to 20 micrograms/kg/min failed to lower BP significantly. The final two trials were preceded by furosemide administration the previous day to reduce fluid volume. Plasma renin activity was low on all three dates. On nephrectomy, the allograft was noted grossly to be infarcted. Histologic examination revealed cortical necrosis, markedly narrowed or occluded intrarenal vessels, and extremely narrowed large vessels within the renal pelvis. The allograft renal artery was thickened and narrowed. From our structural and functional analysis we conclude that (1) hypertension, in this case, was probably volume dependent and was clearly renin independent; and (2) the low renin levels are explainable on the basis of extensive vascular occlusion producing renal infarction and resulting in a kidney incapable of producing significant amounts of renin (autonephrectomy).

Seventy fresh frozen biopsies of 22 human heart allografts were stained with mouse antihuman monoclonal antibodies (OKT-4, OKT-8, and OKM-1) using the immunoperoxidase method. The numbers of infiltrating cell phenotypes were correlated with patients' clinical status and histopathological diagnoses of the biopsies. At the clinically stable stage the number of OKT-4-positive cells (T-4 cells, helper/inducer), OKT-8-positive cells (T-8 cells, suppressor/cytotoxic), OKM-1-positive cells (M-1 cells, monocyte/macrophage) and T4/T8 ratio were lowest. During the early stage of rejection T-4 cells increased to the highest values. T-8 cells also increased significantly and T4/T8 ratio increased to the peak level as well. During the later stage of rejection, T-8 and M-1 cells increased to the highest values and T-4 cells and T4/T8 ratio decreased. After the treatment of rejection T-4 cells continued to decrease and T-8 cells and M-1 cells decreased to intermediate levels, but T4/T8 ratio still remained level. The numbers of T-4 cells, T-8, cells and M-1 cells were closely associated with the histopathologic severity of rejection. These results were also correlated with the allografts' prognoses. Interestingly, high T4/8 ratios with high number of T-4 cells in biopsies during the quiescent period were often followed by rejection episodes within 7 days, even though the pathological diagnoses were mild rejection. Another important finding was that after the treatment of rejection, persistent M-1 cells and low T4/T8 ratios in situ were frequently accompanied by recurrent rejections. Thus, monitoring of infiltrating cell phenotypes may be beneficial in the management of clinical cardiac transplantations.

Cyclosporine is a new immunosuppressive drug that acts early in the exposure of a host to allogeneic stimulation. It is a peptide of fungal origin. It has selective action on T cells, leaving the other cells of the immune system intact. It acts by preventing the function of the early activation signals of T cells, such as the acquisition of receptors for Il 2 and Il 1. It is lipophilic, moderately well absorbed by the gut, and metabolized by the liver. Factors affecting absorption or hepatic metabolism alter the amount of cyclosporine available in the circulation. Circulating levels can be measured by radioimmunoassay or HPLC. Doses should be tailored to trough levels taken approximately 12 hours after an oral or intravenous dose or to individual pharmacokinetic curves. The drug is nephrotoxic, hepatotoxic, and neurotoxic. In addition, cyclosporine has been associated with hypertension, hemolytic-uremic syndrome, increased incidence of intravascular thrombotic events, hypertrichosis, gum hyperplasia, pericardial effusion, and lymphoproliferative disorders. Despite these complications, cyclosporine usage seems to have improved short-term cardiac allograft survival and to have reduced the complications associated with side effects of steroids. As a result, cyclosporine has spawned a resurgence of interest in cardiac transplantation, which will be of great benefit in prolonging the lives of patients with end-stage cardiac disease.

Increased urinary excretion of immunoreactive thromboxane B2 (iTxB2) was found to have a high predictive value, with high sensitivity as an indicator of cardiac allograft rejection in both the immunosuppressed and nonimmunosuppressed rat. In the animals receiving an allograft, urinary iTxB2 excretion significantly increased prior to the onset of rejection, remained elevated, and returned to basal values following completion of the episode. Urinary iTxB2 remained at baseline values in the control animals. The association between rejection and iTxB2 excretion was preserved regardless of the presence or nature of immunosuppression. Urinary iTxB2 excretion increased significantly prior to the reduction of graft beat or histological evidence of rejection. Evaluation of urinary iTxB2 monitoring as a noninvasive indicator for surveillance of clinical cardiac allograft rejection appears to be warranted.