Immunological disruption of antiangiogenic signals by recruited allospecific T cells leads to corneal allograft rejection

Corneal transplantation is the most common solid organ transplantation. The immunologically privileged nature of the cornea results in high success rates. However, T cell-mediated rejection is the most common cause of corneal graft failure. Using antiangiogenesis treatment to prevent corneal neovascularization, which revokes immune privilege, prevents corneal allograft rejection. Endostatin is an antiangiogenic factor that maintains corneal avascularity. In this study, we directly test the role of antiangiogenic and immunological signals in corneal allograft survival, specifically the potential correlation of endostatin production and T cell recruitment. We report that 75% of the corneal allografts of BALB/c mice rejected after postoperative day (POD) 20, whereas all syngeneic grafts survived through POD60. This correlates with endogenous endostatin, which increased and remained high in syngeneic grafts but decreased after POD10 in allografts. Immunostaining demonstrated that early recruitment of allospecific T cells into allografts around POD10 correlated with decreased endostatin production. In Rag(-/-) mice, both allogeneic and syngeneic corneal grafts survived; endostatin remained high throughout. However, after T cell transfer, the allografts eventually rejected, and endostatin decreased. Furthermore, exogenous endostatin treatment delayed allograft rejection and promoted survival secondary to angiogenesis inhibition. Our results suggest that endostatin plays an important role in corneal allograft survival by inhibiting neovascularization and that early recruitment of allospecific T cells into the grafts promotes destruction of endostatin-producing cells, resulting in corneal neovascularization, massive infiltration of effector T cells, and ultimately graft rejection. Therefore, combined antiangiogenesis and immune suppression will be more effective in maintaining corneal allograft survival.     

Mice with Th2-biased immune systems accept orthotopic corneal allografts placed in "high risk" eyes

CD4+ T cells of the Th1 type play a central role in acute rejection of solid tissue grafts, including orthotopic corneal allografts. Th1 cells, which mediate delayed hypersensitivity, are the polar opposites of CD4+ Th2 cells, and the latter cells cross-regulate Th1 cells through the unique pattern of cytokines they secrete. As such, Th2 cells may have a useful role to play in preventing rejection of corneal allografts. To test this possibility, the immune systems of adult mice were biased toward Th2 responses by immunization with keyhole limpet hemocyanin plus IFA. When immunized subsequently with either OVA or allogeneic corneal tissue, these mice acquired Ag-specific primed T cells of the Th2 type. More important, allogeneic corneas grafted into neovascularized eyes of Th2-biased mice experienced significantly enhanced survival. To demonstrate that enhanced survival was promoted by donor-specific Th2 cells, lymphoid cells from keyhole limpet hemocyanin-immune mice bearing healthy corneal allografts suppressed orthotopic corneal allograft rejection when adoptively transferred into naive, syngeneic recipients. We conclude that acceptance of corneal allografts in neovascularized mouse eyes can be significantly enhanced by biasing the recipient immune system toward Th2 responses.     

Cutting edge: atopy promotes Th2 responses to alloantigens and increases the incidence and tempo of corneal allograft rejection

A large body of evidence suggests that corneal allograft rejection is mediated by a type 1 Th cell response and that deviation toward type 2 immunity favors graft survival. However, clinical observations indicate that patients with severe ocular allergies have increased risk of corneal allograft rejection. We used a mouse model of atopic conjunctivitis to evaluate the effects of Th2 immune deviation on corneal allograft survival and possible mechanisms of graft rejection. Our results reveal the following novel findings: 1) atopic conjunctivitis promotes systemic Th2 immune responses to corneal graft donor alloantigens; 2) corneal allografts in atopic host eyes have an increased incidence and swifter tempo of rejection; 3) increased rejection is associated with alterations in systemic T cell-mediated responses to donor alloantigens; and 4) corneal allograft rejection in atopic hosts does not require the direct involvement of infiltrating eosinophils.     

Cutting edge: perforin down-regulates CD4 and CD8 T cell-mediated immune responses to a transplanted organ

Perforin mediates target cell apoptosis by CTLs and NK cells. Although perforin expression correlates strongly with acute allograft rejection, perforin-deficient mice reject allografts with the same kinetics as wild-type recipients. In this study, we tested the hypothesis that while perforin is dispensable for acute rejection, it is essential for down-regulating the alloimmune response by inducing the apoptosis of host immune cells. Using a skin transplantation model, we found that perforin-deficient mice are resistant to the induction of allograft acceptance by agents that block T cell costimulation. Failure to induce allograft acceptance in these mice was observed irrespective of whether the alloimmune response was CD4 or CD8 T cell-mediated and could be attributed to defective apoptosis of activated CD4 and CD8 T cells. In contrast, perforin did not influence T cell proliferation. Therefore, perforin is an essential immunoregulatory molecule that may be required for the induction of transplantation tolerance.     

An age-specific CD8+ T cell pathway that impairs the effectiveness of strategies to prolong allograft survival

Age-related decline in immunity can impair cell-mediated responses during an infection, malignancy, and acute allograft rejection. Although much research has been allocated to understand the immune responses that impact the former two conditions, the cellular mechanisms by which aging impacts the immune acceptance of organ allografts are not completely clear. In this study, we examined how recipient age impacts the efficacy of therapies that modulate immune recognition of allografts using an immunogenic murine skin transplant model. We found that costimulatory blockade-based treatment failed to extend allograft survival in older recipients to the same extent as that observed in younger recipients. CD8(+) T cells were critical for the inability of aged recipients to achieve maximal allograft survival. Although aged mice displayed a larger number of effector memory T cells prior to transplantation, these cells did not exhibit enhanced alloreactivity compared with young memory T cells. In contrast, naive aged CD8(+) T cells exhibited enhanced IFN-γ production to allostimulation compared with young naive T cells. Our results provide evidence that aging enhances CD8(+) T cell alloreactivity. This could impair the ability of costimulatory blockade-based therapies to prolong allograft survival. Thus, targeting CD8(+) T cells in humans may be a way to improve outcomes in older patients requiring immune modulatory therapy.     

The innate NK cells, allograft rejection, and a key role for IL-15

Transplant rejection is mediated primarily by adaptive immune cells such as T cells and B cells. The T and B cells are also responsible for the specificity and memory of the rejection response. However, destruction of allografts involves many other cell types including cells in the innate immune system. As the innate immune cells do not express germline-encoded cell surface receptors that directly recognize foreign Ags, these cells are thought to be recruited by T cells to participate in the rejection response. In this study, we examined the alloreactivity of the innate NK cells in Rag(-/-) mice using a stringent skin transplant model and found that NK cells at a resting state readily reject allogeneic cells, but not the skin allografts. We also found that IL-15, when preconjugated to its high affinity IL-15Ralpha-chain, is remarkably potent in stimulating NK cells in vivo, and NK cells stimulated by IL-15 express an activated phenotype and are surprisingly potent in mediating acute skin allograft rejection in the absence of any adaptive immune cells. Furthermore, NK cell-mediated graft rejection does not show features of memory responses. Our data demonstrate that NK cells are potent alloreactive cells when fully activated and differentiated under certain conditions. This finding may have important clinical implications in models of transplantation and autoimmunity.     

Prevention of allograft tolerance by bacterial infection with Listeria monocytogenes

Exposure to certain viruses and parasites has been shown to prevent the induction of transplantation tolerance in mice via the generation of cross-reactive memory T cell responses or the induction of bystander activation. Bacterial infections are common in the perioperative period of solid organ allograft recipients in the clinic, and correlations between bacterial infections and acute allograft rejection have been reported. However, whether bacterial infections at the time of transplantation have any effect on the generation of transplantation tolerance remains to be established. We used the Gram-positive intracellular bacterium Listeria monocytogenes (LM) as a model pathogen because its effects on immune responses are well described. Perioperative LM infection prevented cardiac and skin allograft acceptance induced by anti-CD154 and donor-specific transfusion in mice. LM-mediated rejection was not due to the generation of cross-reactive T cells and was largely independent of signaling via MyD88, an adaptor for most TLRs, IL-1, and IL-18. Instead, transplant rejection following LM infection was dependent on the expression of the phagosome-lysing pore former listeriolysin O and on type I IFN receptor signaling. Our results indicate that bacterial exposure at the time of transplantation can antagonize tolerogenic regimens by enhancing alloantigen-specific immune responses independently of the generation of cross-reactive memory T cells.     

Donor IFN-gamma receptors are critical for acute CD4(+) T cell-mediated cardiac allograft rejection.

Recent studies using mouse models demonstrate that CD4(+) T cells are sufficient to mediate acute cardiac allograft rejection in the absence of CD8(+) T cells and B cells. However, the mechanistic basis of CD4-mediated rejection is unclear. One potential mechanism of CD4-mediated rejection is via elaboration of proinflammatory cytokines such as IFN-gamma. To determine whether IFN-gamma is a critical cytokine in CD4-mediated acute cardiac allograft rejection, we studied whether the expression of IFN-gamma receptors on the donor heart was required for CD4-mediated rejection. To investigate this possibility, purified CD4(+) T cells were transferred into immune-deficient mice bearing heterotopic cardiac allografts from IFN-gamma receptor-deficient (GRKO) donors. While CD4(+) T cells triggered acute rejection of wild-type heart allografts, they failed to trigger rejection of GRKO heart allografts. The impairment in CD4-mediated rejection of GRKO hearts appeared to primarily involve the efferent phase of the immune response. This conclusion was based on the findings that GRKO stimulator cells provoked normal CD4 proliferation in vitro and that intentional in vivo challenge of CD4 cells with wild-type donor APC or the adoptive transfer of in vitro primed CD4 T cells failed to provoke acute rejection of GRKO allografts. In contrast, unseparated lymph node cells acutely rejected both GRKO and wild-type hearts with similar time courses, illustrating the existence of both IFN-gamma-dependent and IFN-gamma-independent mechanisms of acute allograft rejection.     

B7-H1-induced apoptosis as a mechanism of immune privilege of corneal allografts

The programmed death-1 (PD-1) costimulatory pathway has been demonstrated to play a role in the regulation of immune responses and peripheral tolerance. We investigated the role of this pathway in establishing an immune privilege status of corneal allografts in mice. B7-H1, but not B7-DC or PD-1, was expressed constitutively in the eye, i.e., cornea, iris-ciliary body, and retina. After corneal allografting, PD-1(+)CD4(+) T cells infiltrated and adhered with B7-H1(+) corneal endothelium. Blockade of PD-1 or B7-H1, but not B7-DC, led to accelerated corneal allograft rejection. In B7-H1-expressing corneal allografts, apoptosis of the infiltrating PD-1(+)CD4(+) or CD8(+) T cells was observed, after which there was allograft acceptance. In contrast, B7-H1 blockade suppressed apoptosis of infiltrating PD-1(+) T cells, which led to allograft rejection. In vitro, destruction of corneal endothelial cells by alloreactive T cells was enhanced when the cornea was pretreated with anti-B7-H1 Ab. This is the first demonstration that the constitutive expression of B7-H1 plays a critical role in corneal allograft survival. B7-H1 expressed on corneal endothelial cells maintains long-term acceptance of the corneal allografts by inducing apoptosis of effector T cells within the cornea.     

Testicular immune privilege promotes transplantation tolerance by altering the balance between memory and regulatory T cells

Immune responses are suppressed in immunologically privileged sites, which may provide a unique opportunity to prolong allograft survival. However, it is unknown whether testicular immune privilege promotes transplantation tolerance. Mechanisms underlying immune privilege are also not well understood. Here we found that islet transplantation in the testis, an immunologically privileged site, generates much less memory CD8(+) T cells but induces more Ag-specific CD4(+)CD25(+) regulatory T cells than in a conventional site. These CD4(+)CD25(+) cells exhibited the suppression of alloimmune responses in vivo and in vitro. Despite the immune regulation, intratesticular islet allografts all were rejected within 42 days after transplantation although they survived longer than renal subcapsular islet allografts. However, blocking CD40/CD40L costimulation induced the tolerance of intratesticular, but not renal subcapsular, islet allografts. Tolerance to intratesticular islet allografts spread to skin allografts in the non-privileged sites. Either transfer of memory CD8(+) T cells or deletion of CD25(+) T cells in vivo broke islet allograft tolerance. Thus, transplantation tolerance requires both costimulatory blockade, which suppresses acute allograft rejection, and a favorable balance between memory and regulatory T cells that could favorably prevent late allograft failure. These findings reveal novel mechanisms of immune privilege and provide direct evidence that testicular immune privilege fosters the induction of transplantation tolerance to allografts in both immunologically privileged and non-privileged sites.     

Vascularized bone allograft transplantation in a genetically defined rat model

A heterotopic subcutaneous model for experimental vascularized bone allograft transplantation has been presented. This model uses genetically defined rats and allows serial assessment of graft viability. The reliability of this model has been proven by successful isograft transplantation. This model was used to study the effect of matching at the major histocompatibility complex on vascularized bone allograft survival. Whereas grafts transplanted across a minor histocompatibility barrier survived until sacrifice, grafts transplanted across a major histocompatibility barrier were victims of an acute rejection process. This study, therefore, showed genetic disparity to be a critical determinant of vascularized bone allograft survival. It indicates that primary vascularized bone allografts are as susceptible to rejection as heart and kidney allografts. For these reasons, it can be anticipated that genetic matching will be important in clinical vascularized bone allograft transplantation. The model used in this study should be useful for obtaining further fundamental immunologic information concerning vascularized bone allograft transplantation.     

Roles of deletion and regulation in creating mixed chimerism and allograft tolerance using a nonlymphoablative irradiation-free protocol

The induction of mixed chimerism (MC) is a powerful and effective means to achieve transplantation tolerance in rodent models. Host conditioning with irradiation or cytotoxic drugs has been used in many protocols for chimeric induction across allogeneic barriers. The deletion of alloreactive T cell clones has been described as the main mechanism responsible for the induction of a stable MC. In this study, we demonstrate that a stable MC and skin allograft tolerance can be established across MHC barriers by a noncytotoxic, irradiation-free approach using costimulation blockade plus rapamycin treatment. By using an adoptive transfer model of skin allograft and using specific Vbeta TCR probes, we demonstrated that deletion of donor-reactive cytopathic T cell clones is indeed profound in tolerant hosts. Nonetheless, the challenge of tolerant mixed chimeras with 5 million mononuclear leukocytes (MNL) from naive syngeneic mice was neither able to abolish the stable MC nor to trigger skin allograft rejection, a hallmark of peripheral, not central tolerance. Furthermore, in an adoptive transfer model, MNLs harvested from tolerant hosts significantly inhibited the capacity of naive MNLs to reject same donor, but not third-party, skin allografts. Moreover, when we transplanted skin allografts from stable tolerant chimeras onto syngeneic immune-incompetent mice, graft-infiltrating T cells migrated from the graft site, expanded in the new host, and protected allografts from acute rejection by naive syngeneic MNLs. In this model, both deletional and immunoregulatory mechanisms are active during the induction and/or maintenance of allograft tolerance through creation of MC using a potentially clinically applicable regimen.     

Renal urokinase-type plasminogen activator (uPA) receptor but not uPA deficiency strongly attenuates ischemia reperfusion injury and acute kidney allograft rejection

Central mechanisms leading to ischemia induced allograft rejection are apoptosis and inflammation, processes highly regulated by the urokinase-type plasminogen activator (uPA) and its specific receptor (uPAR). Recently, up-regulation of uPA and uPAR has been shown to correlate with allograft rejection in human biopsies. However, the causal connection of uPA/uPAR in mediating transplant rejection and underlying molecular mechanisms remain poorly understood. In this study, we evaluated the role of uPA/uPAR in a mice model for kidney ischemia reperfusion (IR) injury and for acute kidney allograft rejection. uPAR but not uPA deficiency protected from IR injury. In the allogenic kidney transplant model, uPAR but not uPA deficiency of the allograft caused superior recipient survival and strongly attenuated loss of renal function. uPAR-deficient allografts showed reduced generation of reactive oxygen species and apoptosis. Moreover, neutrophil and monocyte/macrophage infiltration was strongly attenuated and up-regulation of the adhesion molecule ICAM-1 was completely abrogated in uPAR-deficient allografts. Inadequate ICAM-1 up-regulation in uPAR(-/-) primary aortic endothelial cells after C5a and TNF-alpha stimulation was confirmed by in vitro experiments. Our results demonstrate that the local renal uPAR plays an important role in the apoptotic and inflammatory responses mediating IR-injury and transplant rejection.     

Pretransplant frequency of donor-specific, IFN-gamma-producing lymphocytes is a manifestation of immunologic memory and correlates with the risk of posttransplant rejection episodes.

While matching for MHC Ags improves renal allograft survival, closely matched grafts sometimes fail due to rejection, and poorly matched allografts are often well tolerated by the recipient. The severity of the rejection process may partially depend on the presence of environmentally primed T cells in the recipient that cross-react with donor Ags. To test for the presence of primed, donor-specific T cells in humans before transplantation, we used an enzyme-linked immunospot assay for detection of allospecific cytokines produced by individual human PBLs. We demonstrate that this approach detects cytokine production at single cell resolution and detects production of IFN-gamma only when there is defined immunologic priming, thus representing a measure of primed donor-specific immunity. Because the environmental Ag exposure of the recipient is not a function of the HLA mismatch between donor and potential recipient, the number of HLA mismatches may not correlate with the frequency of pretransplant, donor-specific IFN-gamma-producing PBLs. Studies of donor-specific IFN-gamma-producing lymphocytes in a cohort of patients being evaluated for renal transplantation corroborated this hypothesis. Moreover, for recipients of both living and cadaver renal allografts, the pretransplant frequency of donor-specific memory cells correlated with the posttransplant risk of developing acute rejection episodes. This improved ability to define the strength of the allospecific immune response by enzyme-linked immunospot assay may allow improved pairing of recipients with donors and identification of kidney allograft donor-recipient pairs at high risk for acute rejection, thus permitting targeted interventions aimed at prolonging graft survival.     

The function of donor versus recipient programmed death-ligand 1 in corneal allograft survival

Programmed death-ligand (PD-L)1 and PD-L2, newer B7 superfamily members, are implicated in the negative regulation of immune responses and peripheral tolerance. To examine their function in alloimmunity, we used the murine model of orthotopic corneal transplantation. We demonstrate that PD-L1, but not PD-L2, is constitutively expressed at high levels by the corneal epithelial cells, and at low levels by corneal CD45+ cells in the stroma, whereas it is undetectable on stromal fibroblasts and corneal endothelial cells. Inflammation induces PD-L1 up-regulation by corneal epithelial cells, and infiltration of significant numbers of PD-L1+CD45+CD11b+ cells. Blockade with anti-PD-L1 mAb dramatically enhances rejection of C57BL/6 corneal allografts by BALB/c recipients. To examine the selective contribution of donor vs host PD-L1 in modulating allorejection, we used PD-L1-/- mice as hosts or donors of combined MHC and minor H-mismatched corneal grafts. BALB/c grafts placed in PD-L1-/- C57BL/6 hosts resulted in pronounced T cell priming in the draining lymph nodes, and universally underwent rapid rejection. Allografts from PD-L1-/- C57BL/6 donors were also significantly more susceptible to rejection than wild-type C57BL/6 grafts placed into BALB/c hosts, primarily as a result of increased T cell infiltration rather than enhanced priming. Taken together, our results identify differential roles for recipient vs donor PD-L1 in regulating induction vs effector of alloimmunity in corneal grafts, the most common form of tissue transplantation, and highlight the importance of peripheral tissue-derived PD-L1 in down-regulating local immune responses.     

Virus-induced abrogation of transplantation tolerance induced by donor-specific transfusion and anti-CD154 antibody

Treatment with a 2-week course of anti-CD154 antibody and a single transfusion of donor leukocytes (a donor-specific transfusion or DST) permits skin allografts to survive for >100 days in thymectomized mice. As clinical trials of this methodology in humans are contemplated, concern has been expressed that viral infection of graft recipients may disrupt tolerance to the allograft. We report that acute infection with lymphocytic choriomeningitis virus (LCMV) induced allograft rejection in mice treated with DST and anti-CD154 antibody if inoculated shortly after transplantation. Isografts resisted LCMV-induced rejection, and the interferon-inducing agent polyinosinic:polycytidylic acid did not induce allograft rejection, suggesting that the effect of LCMV is not simply a consequence of nonspecific inflammation. Administration of anti-CD8 antibody to engrafted mice delayed LCMV-induced allograft rejection. Pichinde virus also induced acute allograft rejection, but murine cytomegalovirus and vaccinia virus (VV) did not. Injection of LCMV approximately 50 days after tolerance induction and transplantation had minimal effect on subsequent allograft survival. Treatment with DST and anti-CD154 antibody did not interfere with clearance of LCMV, but a normally nonlethal high dose of VV during tolerance induction and transplantation killed graft recipients. We conclude that DST and anti-CD154 antibody induce a tolerant state that can be broken shortly after transplantation by certain viral infections. Clinical application of transplantation tolerance protocols may require patient isolation to facilitate the procedure and to protect recipients.     

CD4+ T lymphocytes are not necessary for the acute rejection of vascularized mouse lung transplants

Acute rejection continues to present a major obstacle to successful lung transplantation. Although CD4(+) T lymphocytes are critical for the rejection of some solid organ grafts, the role of CD4(+) T cells in the rejection of lung allografts is largely unknown. In this study, we demonstrate in a novel model of orthotopic vascularized mouse lung transplantation that acute rejection of lung allografts is independent of CD4(+) T cell-mediated allorecognition pathways. CD4(+) T cell-independent rejection occurs in the absence of donor-derived graft-resident hematopoietic APCs. Furthermore, blockade of the CD28/B7 costimulatory pathways attenuates acute lung allograft rejection in the absence of CD4(+) T cells, but does not delay acute rejection when CD4(+) T cells are present. Our results provide new mechanistic insight into the acute rejection of lung allografts and highlight the importance of identifying differences in pathways that regulate the rejection of various organs.     

Orthotopic Hind Limb Transplantation in the Mouse

In vivo animal model systems, and in particular mouse models, have evolved into powerful and versatile scientific tools indispensable to basic and translational research in the field of transplantation medicine. A vast array of reagents is available exclusively in this setting, including mono- and polyclonal antibodies for both diagnostic and interventional applications. In addition, a vast number of genotyped, inbred, transgenic, and knock out strains allow detailed investigation of the individual contributions of humoral and cellular components to the complex interplay of an immune response and make the mouse the gold standard for immunological research. Vascularized Composite Allotransplantation (VCA) delineates a novel field of transplantation using allografts to replace "like with like" in patients suffering traumatic or congenital tissue loss. This surgical methodological protocol shows the use of a non-suture cuff technique for super-microvascular anastomosis in an orthotopic mouse hind limb transplantation model. The model specifically allows for comparison between established paradigms in solid organ transplantation with a novel form of transplants consisting of various different tissue components. Uniquely, this model allows for the transplantation of a viable vascularized bone marrow compartment and niche that have the potential to exert a beneficial effect on the balance of immune acceptance and rejection. This technique provides a tool to investigate alloantigen recognition and allograft rejection and acceptance, as well as enables the pursuit of functional nerve regeneration studies to further advance this novel field of transplantation.     

Increased expression of Tim-3 and its ligand Galectin-9 in rat allografts during acute rejection episodes

The aim of the study is to elucidate the profiles of T-cell immunoglobulin and mucin domain-3 (Tim-3) and its ligand Galecin-9 in acute pulmonary rejection by using a rat model of lung transplantation. Left lung grafts retrieved from Lewis or Fisher 344 rats were orthotopically transplanted into Lewis recipients without any immunosuppressions; the grafts were harvested at day 3, 7 or 10 after transplantation. The grade of acute rejection was histopathologically evaluated. Tim-3, Galectin-9, immune antigen and related cytokines expression were assessed with immunological techniques and real-time polymerase chain reaction (RT-PCR), respectively. Then, our results showed that Tim-3 and its ligand Galectin-9 were markedly up-regulated at protein and mRNA levels in allografts compared with syngrafts. Meanwhile, the decreased CD4/CD8 ratio was associated with acute rejection occurring and Tim-3 expression on CD4⁺ and CD8⁺ T cells in allografts was increased. Therefore, our study firstly described that enhanced Tim-3 and its ligand Galectin-9 in allografts might play an important role in the pathogenesis of rat lung transplant rejection, implying new valuable markers for detecting acute allograft rejection.