Antibody immunosuppressive therapy in solid organ transplant: Part II

The use of antibodies in transplantation dates back to 1986 when muromonab CD3, a monoclonal antibody (mAb) targeting CD3, was first approved for prevention and treatment of renal allograft rejection. These agents have largely been used in a brief adjunctive manner to provide immunosuppression during the initial period after solid organ transplantation or during an episode of acute rejection. Recent advances in our understanding of transplant immunology have allowed emergence of numerous new mAbs, targeting co-stimulatory signals, cell surface receptors and novel protein constructs. During the next decade, transplant professionals will increasingly require knowledge of the mechanisms and pharmacologic characteristics of these novel therapeutic agents.Key words: antibody, immunosuppression, transplantation, biologics, anti-adhesion     

Design,synthesis, and evaluation of 4,6-diaminonicotinamide derivatives as novel and potent immunomodulators targeting JAK3

In organ transplantation, T cell-mediated immune responses play a key role in the rejection of allografts. Janus kinase 3 (JAK3) is specifically expressed in hematopoietic cells and associated with regulation of T cell development via interleukin-2 signaling pathway. Here, we designed novel 4,6-diaminonicotinamide derivatives as immunomodulators targeting JAK3 for prevention of transplant rejection. Our optimization of C4- and C6-substituents and docking calculations to JAK3 protein confirmed that the 4,6-diaminonicotinamide scaffold resulted in potent inhibition of JAK3. We also investigated avoidance of human ether-a-go-go related gene (hERG) inhibitory activity. Selected compound 28 in combination with tacrolimus prevented allograft rejection in a rat heterotopic cardiac transplantation model.     

Increased Numbers of Circulating CD8 Effector Memory T Cells before Transplantation Enhance the Risk of Acute Rejection in Lung Transplant Recipients

The effector and regulatory T cell subpopulations involved in the development of acute rejection episodes in lung transplantation remain to be elucidated. Twenty-seven lung transplant candidates were prospectively monitored before transplantation and within the first year post-transplantation. Regulatory, Th17, memory and naïve T cells were measured in peripheral blood of lung transplant recipients by flow cytometry. No association of acute rejection with number of peripheral regulatory T cells and Th17 cells was found. However, effector memory subsets in acute rejection patients were increased during the first two months post-transplant. Interestingly, patients waiting for lung transplant with levels of CD8⁺ effector memory T cells over 185 cells/mm³ had a significant increased risk of rejection [OR: 5.62 (95% CI: 1.08-29.37), p=0.04]. In multivariate analysis adjusted for age and gender the odds ratio for rejection was: OR: 5.89 (95% CI: 1.08-32.24), p=0.04. These data suggest a correlation between acute rejection and effector memory T cells in lung transplant recipients. The measurement of peripheral blood CD8⁺ effector memory T cells prior to lung transplant may define patients at high risk of acute lung rejection.     

Both CD4(+)CD25(+) and CD4(+)CD25(-) regulatory cells mediate dominant transplantation tolerance

CD4(+)CD25(+) T cells have been proposed as the principal regulators of both self-tolerance and transplantation tolerance. Although CD4(+)CD25(+) T cells do have a suppressive role in transplantation tolerance, so do CD4(+)CD25(-) T cells, although 10-fold less potent. Abs to CTLA-4, CD25, IL-10, and IL-4 were unable to abrogate suppression mediated by tolerant spleen cells so excluding any of these molecules as critical agents of suppression. CD4(+)CD25(+) T cells from naive mice can also prevent rejection despite the lack of any previous experience of donor alloantigens. However, this requires many more naive than tolerized cells to provide the same degree of suppression. This suggests that a capacity to regulate transplant rejection pre-exists in naive mice, and may be amplified in "tolerized" mice. Serial analysis of gene expression confirmed that cells sorted into CD4(+)CD25(+) and CD4(+)CD25(-) populations were distinct in that they responded to TCR ligation with very different programs of gene expression. Further characterization of the differentially expressed genes may lead to the development of diagnostic tests to monitor the tolerant state.     

Molecular mechanisms of immunity in corneal allotransplantation and xenotransplantation

Corneal allotransplantation is the most common and successful form of solid organ transplantation in humans. In uncomplicated cases, the two-year graft survival rate is over 90%. This extraordinary success can be attributed in part to various features of the normal cornea and anterior segment that together account for their 'immune-privileged' status. However, despite this success, a significant number of corneal grafts fail and immunological rejection remains by far the leading cause of graft failure. Studies on animal models of corneal transplantation have yielded a wealth of information on the molecular and cellular features of graft rejection, and have established that this process is mediated primarily by CD4+ T cells of the T helper 1 (Th1) phenotype. In addition, studies have elucidated that certain facets of allosensitisation differ between corneal and other solid organ transplants. On the basis of these findings, novel experimental strategies selectively targeting the afferent or efferent arms of corneal alloimmunity have provided promising results in preventing corneal allograft rejection in the laboratory. Finally, because of the global shortage of human donor corneas, there is currently renewed interest in the possibility of using corneas from other species for transplantation into human eyes (xenotransplantation). Preliminary studies on animal models of corneal xenotransplantation have documented both antibody-mediated and cell-mediated responses that might play important roles in the accelerated rejection observed in corneal xenotransplants. This review synthesises the principal concepts emerging from studies of the molecular mechanisms in corneal transplant immunology.     

Inhibiting CXCR3-dependent CD8+ T cell trafficking enhances tolerance induction in a mouse model of lung rejection

Lung transplantation remains the only effective therapy for patients with end-stage pulmonary diseases. Unfortunately, acute rejection of the lung remains a frequent complication and is an important cause of morbidity and mortality. The induction of transplant tolerance is thought to be dependent, in part, on the balance between allograft effector mechanisms mediated by effector T lymphocytes (Teff), and regulatory mechanisms mediated by FOXP3(+) regulatory T cells (Treg). In this study, we explored an approach to tip the balance in favor of regulatory mechanisms by modulating chemokine activity. We demonstrate in an adoptive transfer model of lung rejection that CXCR3-deficient CD8(+) Teff have impaired migration into the lungs compared with wild-type Teff, which results in a dramatic reduction in fatal pulmonary inflammation. The lungs of surviving mice contained tolerized CXCR3-deficient Teff, as well as a large increase in Treg. We confirmed that Treg were needed for tolerance and that their ability to induce tolerance was dependent on their numbers in the lung relative to the numbers of Teff. These data suggest that transplantation tolerance can be achieved by reducing the recruitment of some, but not necessarily all, CD8(+) Teff into the target organ and suggest a novel approach to achieve transplant tolerance.     

DR(high+)CD45RA(-)-Tregs potentially affect the suppressive activity of the total Treg pool in renal transplant patients

Recent studies show that regulatory T cells (Tregs) play an essential role in tolerance induction after organ transplantation. In order to examine whether there are differences in the composition of the total CD4(+)CD127(low+/-)FoxP3(+)- Treg cell pool between stable transplant patients and patients with biopsy proven rejection (BPR), we compared the percentages and the functional activity of the different Treg cell subsets (DR(high+)CD45RA(-)-Tregs, DR(low+)CD45RA(-)-Tregs, DR(-)CD45RA(-)-Tregs, DR(-)CD45RA(+)-Tregs). All parameters were determined during the three different periods of time after transplantation (0-30 days, 31-1,000 days, >1,000 days). Among 156 transplant patients, 37 patients suffered from BPR. The most prominent differences between rejecting and non-rejecting patients were observed regarding the DR(high+)CD45RA(-)-Treg cell subset. Our data demonstrate that the suppressive activity of the total Treg pool strongly depends on the presence of these Treg cells. Their percentage within the total Treg pool strongly decreased after transplantation and remained relatively low during the first year after transplantation in all patients. Subsequently, the proportion of this Treg subset increased again in patients who accepted the transplant and reached a value of healthy non-transplanted subjects. By contrast, in patients with acute kidney rejection, the DR(high+)CD45RA(-)-Treg subset disappeared excessively, causing a reduction in the suppressive activity of the total Treg pool. Therefore, both the monitoring of its percentage within the total Treg pool and the monitoring of the HLA-DR MFI of the DR(+)CD45RA(-)-Treg subset may be useful tools for the prediction of graft rejection.     

Current status of renal transplantation.

The success rate of renal transplantation has improved considerably during the past decade, with substantial improvements in both graft and patient survival. The quality of graft function, however, and not graft survival alone is increasingly determining the standards by which transplantation outcome is being judged. As the demand for kidney transplants continues to rise and transplants are being offered to an ever-increasing number of patients, organs are being sought from new supply pools and efforts are being made to use current resources more efficiently. Improvements in clinical management have allowed short-term complications such as infection and rejection to be better prevented or better diagnosed and treated. Fundamental advances in the understanding of the immunologic processes underlying both allograft rejection and acceptance and the introduction of new immunosuppressive agents have allowed a better use of drug therapy and have moved the goal of acquired transplant tolerance closer to attainment. With improved initial transplant success rates, the long-term transplantation outcome is becoming more important. The role of tissue matching in preventing chronic rejection is becoming more appreciated, and the long-term risks of malignancy, arteriosclerosis, and chronic rejection are being better recognized and managed.     

Modulation of tissue-specific immune response to cardiac myosin can prolong survival of allogeneic heart transplants

The role of immune response to tissue-specific Ags in transplant rejection is poorly defined. We have previously reported that transplantation of cardiac allografts triggers a CD4(+) Th1 cell response to cardiac myosin (CM), a major contractile protein of the heart, and that pretransplant activation of proinflammatory CM-specific T cells accelerates rejection. In this study, we show that administration of CM together with IFA (CM/IFA) can prevent acute rejection of an allogeneic heart transplant. Prolongation of cardiac graft survival is associated with activation of CM- and allo-specific T cells secreting type 2 cytokines (IL-4, IL-5) and reduction of the frequency of proinflammatory IFN-gamma-secreting (type 1) alloreactive T cells. Blocking of IL-4 cytokine with Abs abrogates the prolongation. CM/IFA treatment prevents acute rejection of MHC class I-mismatched, but not fully mismatched grafts. However, if donor heart is devoid of MHC class II expression, CM-IFA administration delays rejection of fully allogeneic cardiac transplants. This finding suggests that the effect of CM modulation depends on the type (direct vs indirect) and strength of recipient's CD4(+) T cell alloresponse. Our results underscore the important role of host immunity to tissue-specific Ags in the rejection of an allograft. This study demonstrates that modulation of the immune response to a tissue-specific Ag can significantly prolong cardiac allograft survival, an observation that may have important implications for the development of novel selective immune therapies in transplantation.     

CD8 T cells specific for a donor-derived, self-restricted transplant antigen are nonpathogenic bystanders after vascularized heart transplantation in mice

CD8 T cell cross-priming, an established mechanism of protective antiviral immunity, was originally discovered during studies involving minor transplantation Ags. It is unclear whether or how cross-primed CD8 T cells, reactive to donor-derived, but recipient class I MHC-restricted epitopes, could injure a fully MHC-disparate, vascularized transplant. To address this question we studied host class I MHC-restricted, male transplantation Ag-reactive T cell responses in female recipients of fully MHC-disparate, male heart transplants. Cross-priming to the immune-dominant determinant HYUtyp occurred at low frequency after heart transplantation. CD8 T cell preactivation through immunization with HYUtyp mixed in CFA did not alter the kinetics of acute rejection. Furthermore, neither HYUtyp immunization nor adoptive transfer of HYUtyp-specific TCR-transgenic T cells affected outcome in 1) a model of chronic rejection in the absence of immunosuppression or 2) a model of allograft acceptance induced by costimulatory blockade. The results support the contention that CD8 T cells reactive to host-restricted, but donor-derived, Ags are highly specific and are nonpathogenic bystanders during rejection of MHC-disparate cardiac allografts.     

Immunomodulatory effects of BXL-01-0029, a less hypercalcemic vitamin D analogue, in human cardiomyocytes and T cells

Current immunosuppressive protocols have reduced rejection occurrence in heart transplantation; nevertheless, management of heart transplant recipients is accompanied by major adverse effects, due to drug doses close to toxic range. In allograft rejection, characterized by T-helper 1 (Th1) cell-mediated response, the CXCL10-CXCR3 axis plays a pivotal role in triggering a self-promoting inflammatory loop. Indeed, CXCL10 intragraft production, required for initiation and development of graft failure, supports organ infiltration by Th1 cells. Thus, targeting the CXCL10-CXCR3 axis while avoiding generalized immunosuppression, may be of therapeutic significance. Based on preclinical evidence for immunoregulatory properties of vitamin D receptor agonists, we propose that a less hypercalcemic vitamin D analogue, BXL-01-0029, might have the potential to contribute to rejection management. We investigated the effect of BXL-01-0029 on CXCL10 secretion induced by proinflammatory stimuli, both in human isolated cardiomyocytes (Hfcm) and purified CD4+ T cells. Mycophenolic acid (MPA), the active agent of mycophenolate mofetil, was used for comparison. BXL-01-0029 inhibited IFNγ and TNFα-induced CXCL10 secretion by Hfcm more potently than MPA, impairing cytokine synergy and pathways. BXL-01-0029 reduced also CXCL10 protein secretion and gene expression by CD4+ T cells. Furthermore, BXL-01-0029 did not exert any toxic effect onto both cell types, suggesting its possible use as a dose-reducing agent for conventional immunosuppressive drugs in clinical 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.     

CD4+ CD45RB low-density cells from untreated mice prevent acute allograft rejection.

In the absence of therapy that suppresses the action of the immune system, the immune response to transplantation Ags results in rapid rejection of the transplant. The most successful mechanism so far described that achieves organ-specific immunological tolerance is that which controls peripheral tolerance to self-tissue. Until now, no similarities have been documented between the peripheral response to self-Ags and the response to transplantation Ags. CD4+ cells that express a high density of CD45RB (in the mouse) and CD45RC (in the rat) on their surface have been shown to cause a number of autoimmune disorders. In contrast, autoimmunity caused by the CD45RB high-density cells is inhibited by CD4+ CD45RB cells that express a low density of CD45RB (CD45RC in the rat). In this paper we show that CD4+ CD45RB high-density cells are sufficient to cause rejection of a MHC-mismatched pancreas allograft, whereas CD4+ CD45RB low-density cells are not. Unexpectedly, the CD45RB low-density cells prevent the CD45RBhigh expressing cells from causing rejection. These data suggest that the response to foreign tissue can be controlled in the same way as the response to self-tissue.     

Targeted contrast agents for magnetic resonance imaging and ultrasound

The development of contrast agents that can be localized to a particular tissue or cellular epitope will potentially allow the noninvasive visualization and characterization of a variety of disease states. Recent advances have been made in the field of molecular imaging with magnetic resonance imaging and ultrasound and varied approaches have been devised to overcome the high background tissue signal. The types of agents and applications developed include gadolinium-conjugated targeting molecules for imaging of fibrin, superparamagnetic iron oxide particles for stem-cell tracking, multimodal perfluorocarbon nanoparticles for visualization of angiogenesis, liposomes for targeting atheroma components, and microbubbles for imaging transplant rejection.     

Comparison of long-term immunosuppression for limb transplantation using cyclosporine, tacrolimus, and mycophenolate mofetil: implications for clinical composite tissue transplantation

This study compared the efficacy of long-term intermittent immunosuppression in preventing the rejection of a limb transplant across the strongest histocompatibility barrier in ACI --> Lewis rats using the conventional immunosuppressive agent cyclosporine-A and the newer immunosuppressive agents FK-506 (tacrolimus) and RS-61443 (mycophenolate mofetil). The recipient animals were immunosuppressed daily for 14 days postoperatively, followed by long-term intermittent, twice-weekly immunosuppression using cyclosporine 25 mg/kg, RS-61443 30 mg/kg, or FK-506 2 mg/kg. All three immunosuppressive agents were able to prolong the rejection of the skin component of a limb transplant compared with nonimmunosuppressed controls. Eight of nine animals receiving cyclosporine immunosuppression showed signs of rejection of the skin component of the limb transplant while continuing to receive long-term immunosuppression and had a mean rejection time of 61.6 days. Seven of 10 animals immunosuppressed with RS-61443 also showed signs of rejection while still receiving immunosuppression, with a mean rejection time of 43.6 days. Nine of 10 animals receiving FK-506 immunosuppression showed no signs of skin rejection, but died of bacterial pneumonia between 273 and 334 days after transplantation, with a mean rejection time of 296.1 days. There was no statistically significant difference between intermittent immunosuppression with cyclosporine and RS-61443, but FK-506 was significantly superior to both cyclosporine and RS-61443. The implication of this study is that FK-506, but not cyclosporine or RS-61443, is probably the only single immunosuppressive agent capable of preventing rejection of the skin component of a composite tissue transplant. Combination immunosuppression with FK-506 and RS-61443, therefore, may be required to allow composite tissue transplantation to become a predictable clinical reality in the future.     

The role of the CD134-CD134 ligand costimulatory pathway in alloimmune responses in vivo

The CD134-CD134 ligand (CD134L) costimulatory pathway has been shown to be critical for both T and B cell activation; however, its role in regulating the alloimmune response remains unexplored. Furthermore, its interactions with other costimulatory pathways and immunosuppressive agents are unclear. We investigated the effect of CD134-CD134L pathway blockade on allograft rejection in fully MHC-mismatched rat cardiac and skin transplantation models. CD134L blockade alone did not prolong graft survival compared with that of untreated recipients, and in combination with donor-specific transfusion, cyclosporine, or rapamycin, was less effective than B7 blockade in prolonging allograft survival. However, in combination with B7 blockade, long-term allograft survival was achieved in all recipients (>200 days). Moreover, this was synergistic in reducing the frequency of IFN-gamma-producing alloreactive lymphocytes and inhibiting the generation of activated/effector lymphocytes. Most impressively, this combination prevented rejection in a presensitized model using adoptive transfer of primed lymphocytes into athymic heart transplant recipients. In comparison to untreated recipients (mean survival time (MST): 5.3 +/- 0.5 days), anti-CD134L mAb alone modestly prolonged allograft survival (MST: 14 +/- 2.8 days) as did CTLA4Ig (MST: 21.5 +/- 1.7 days), but all grafts were rejected within 24 days. Importantly, combined blockade further and significantly prolonged allograft survival (MST: 75.3 +/- 12.7 days) and prevented the expansion and/or persistence of primed/effector alloreactive T cells. Our data suggest that CD134-CD134L is a critical pathway in alloimmune responses, especially recall/primed responses, and is synergistic with CD28-B7 in mediating T cell effector responses during allograft rejection. Understanding the mechanisms of collaboration between these different pathways is important for the development of novel strategies to promote long-term allograft survival.     

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.     

CD70 signaling is critical for CD28-independent CD8+ T cell-mediated alloimmune responses in vivo

The inability to reproducibly induce robust and durable transplant tolerance using CD28-B7 pathway blockade is in part related to the persistence of alloreactive effector/memory CD8(+) T cells that are less dependent on this pathway for their cellular activation. We studied the role of the novel T cell costimulatory pathway, CD27-CD70, in alloimmunity in the presence and absence of CD28-B7 signaling. CD70 blockade prolonged survival of fully mismatched vascularized cardiac allografts in wild-type murine recipients, and in CD28-deficient mice induced long-term survival while significantly preventing the development of chronic allograft vasculopathy. CD70 blockade had little effect on CD4(+) T cell function but prevented CD8(+) T cell-mediated rejection, inhibited the proliferation and activation of effector CD8(+) T cells, and diminished the expansion of effector and memory CD8(+) T cells in vivo. Thus, the CD27-CD70 pathway is critical for CD28-independent effector/memory CD8(+) alloreactive T cell activation in vivo. These novel findings have important implications for the development of transplantation tolerance-inducing strategies in primates and humans, in which CD8(+) T cell depletion is currently mandatory.