A better understanding of the immune system and advances in immunosuppressive medications have reduced the incidence of acute rejection in the first year of transplant from 50% in the 1990s to 10% in 2009 . It was believed that graft damage was caused solely by T cells and not antibodies. In the 1990s, it was demonstrated that the development of antibodies was connected to the antibody-mediated rejection (AMR), and the presence of donor-specific antibodies (DSA) is increasingly being recognized to play a major role in graft dysfunction, longevity, and loss [2▪▪]. Although fewer than 10% of kidney transplant patients experience AMR, as many as 30% of them experience graft loss as a consequence of DSA. The consequent long-term survival in AMR-positive patients is 70% in comparison with 97% in those who are negative .
ALLOANTIBODIES AND HUMORAL REJECTION
DSA are nearly always directed against human leukocyte antigens (HLAs) molecules, and often activate the complement. Less frequently DSA are directed against different antigens, ABO blood group antigens, minor histocompatibility antigens [anti-major histocompatibility factor class 1 related chain A (MICA)], endothelial cell antigens, and angiotensin II type I receptors.
Factors involved in the development of DSA are retransplantation, young age, deceased donor transplantation, pregnancy, nonadherence, insufficient immunosuppression, HLA alloantibodies before transplantation, HLA-DR or HLA-DQ mismatch, inflammation, and T-cell-mediated rejection [4▪▪]. Proteins and stem cells of donor origin persist even after removal of the graft, thus maintaining an immunological stimulus .
Cell damage during the ischemia reperfusion time or during delayed graft function could be another source of antibodies. The grafted necrotic or apoptotic cells could release antigens, which are able to trigger humoral immunity reactions.
De-novo antibodies, mostly anti-HLA, have been detected in up to 24% of children with a renal transplant, as shown in a report from a United States multicenter. In an Italian single-center study, it was shown that out of the 82 children who underwent kidney transplantation without prior DSA, 23% developed de-novo DSA after 4 years of follow-up, which were mostly directed against HLA-DQ antigens. A significant correlation was found between DSA and chronic AMR. The conclusions were that children developing DSA are at risk of graft dysfunction, and that there is the great need of developing new strategies to prevent antibody-mediated graft damage and progression to graft failure. The presence of a persistent large panel of antibodies against HLA and panel reactive antibodies (PRA) greater than 50% in patients who are candidates for a kidney transplant provoke the need for a desensitization approach that increases the chance of receiving a graft . For these reasons, serial monitoring of DSA has been proposed after renal transplantation.
TEST TO DETECT ALLOANTIBODIES AND SPECIFICITY
Recently, two highly sensitive assays have been developed to screen recipients for the number, specificity and binding strength of anti-HLA alloantibodies : the ELISA and the flow cytometry or Luminex assay, of which the latter is more sensitive (Fig. 1). An important, recent innovation is the ability to evaluate alloantibodies as binding complement (C1q).
COMPLEMENT SYSTEM AND ALLOANTIBODIES
Antibodies themselves do not destroy allografted cells, as they need to activate complement or cytotoxic cells.
The activation of complement with the production of the C5–C9 complex (MAC) induces glomerulitis and capillarities. Interestingly, C4d, a split protein, produced during complement activation, can covalently bind to the endothelium or basement membrane collagen, becoming a marker of AMR. In a few cases, DSA can cause endothelial damage via cell-mediated cytotoxicity, complement-independent. This mechanism seems to be more frequent in chronic AMR.
The Banff classification defines three criteria for the diagnosis of AMR: the histologic evidence of acute/chronic tissue injury (glomerulitis, peritubular capillarities), the C4d immunostaining of peritubular capillaries, and the presence of DSA. Microvascular injury is more strongly associated with graft loss than C4d positivity, even if C4d positivity is detectable in the more severe form of AMR.
Some patients that show overexpression of particular endothelial genes (the so-called enodothelial associated transcript), including the gene encoding for vonWF , are prone to losing the graft with an histologic pattern of transplant glomerulopathy, particularly if there is a coinciding presence of high titers of DSA. These cases are generally C4d negative, and the Banff group is now working to define the criteria for these  (Fig. 2).
The severity of AMR increases the need for a targeted therapy, focused on both reducing the synthesis of DSA and removing them from the bloodstream. High doses of steroids are a nonspecific option for AMR and only 50% of the patients have a response with graft survival of 15%–65%.
A better option is the removal of DSA by plasmapheresis/immunoadsorption with or without intravenous immunoglobulins (IVIg). Several protocols have been proposed that could also be used to treat children. The desensitizing protocols include removal of DSA by high-dose IVIg administration, plasmapheresis, immunoadsorption or a combination of these two. The use of plasmapheresis/immunoadsorption, with or without IVIg, showed an 80–90% reversal of AMR and graft survival of 80% at 18 months. This therapy has only limited effect on chronic AMR .
Intravenous immunoglobulin is used at different – but generally high – doses, with different schemes. In addition, the effect of substitution due to the plasmapheresis/immunoadsorption-related losses, the immunoglobulin has an immunomodulatory role on B and T cells, probably by inducing apoptosis of B cells and regulating B-cell signaling. Another effect is that it inhibits binding of DSA to endothelial cells and complement activation. In one study, it was shown that weekly infusion of high-dose (500 mg/kg) IVIg for three consecutive weeks every 12 weeks reduced PRA to zero, and this effect lasted for over 3 years . Another study reported a case in which IVIg was successfully used to reduce PRA from 95% to 15%, allowing retransplantation in a 13-year-old boy . In adults, IVIg alone has not produced satisfactory results.
The purpose of a true targeted therapy, however, is to reduce the synthesis of DSA by affected B, or, more ideally, the long-lived plasma cells. The great majority of these drugs is not approved by the United States Food and Drug Administration (US FDA) and are used only on the basis of their effect on humoral immunity. Examples of these drugs are: Rituximab, Alemtuzumab, Bortezomib, and Eculizumab. Only in desperate cases, when the patient is unresponsive to all other types of treatments, rescue splenectomy has been used for refractory AMR. It should be noted that all cases of AMR are associated with signs of T-cell rejection; therefore, treatment suppressing T-cell activity is mandatory.
Rituximab, an antibody specific for the cluster of differentiation 20 (CD20) receptor on B cells, cannot, by itself, reduce anti-HLA antibody levels, but it can prevent clonal B-cell expansion and, consequently, DSA production. The advantage of Rituximab (1 g/1.73 m2) for children is its high efficacy in treating nephrotic syndrome with low incidence of infections. In some protocols, Rituximab was given after plasmapheresis . In one study, children with active chronic DSA rejection were treated with 4 weekly doses of 1 g/kg IVIg followed by one single dose of Rituximab (375 mg/m2). In four out of six cases, a significantly lower loss of glomerular filtration rate (GFR) over 6 months of treatment was observed . These results were confirmed in a larger trial that included 20 children who were followed up for over 2 years. This study demonstrated a clinical response (evaluated as reduction of GFR loss) in 70% of the patients. Meanwhile, there was a reduction of 60% of antibodies against both HLA class I and class II .
The combination of Rituximab with plasmapheresis was able to maintain the immunoglobulin-depleting effect of plasmapheresis for a longer time, thereby allowing the use of this protocol in deceased donor transplants as well.
The major drawbacks of these protocols are the risk of infections and the rebound of antibodies. This means there is only a short time window for receiving a transplant, and repeated desensitization is required if a suitable donor is not found.
Bortezomib, a proteasome inhibitor capable of eliminating long-lived plasma cells, was first used at Mayo Clinic and demonstrated its superiority when compared to plasmapheresis alone. Nearly 67% of patients treated with it show reduced plasma cells number (bone marrow analysis) compared with none of the patients treated with plasmapheresis alone . Similar results were obtained at the University of Cincinnati .
Another proteasome inhibitor, carfilzomib, is soon to receive FDA approval. It differs from bortezomib in that carfilzomib irreversibly inhibits the proteasome . It has been studied and shown to be effective in treating bortezomib-refractory myeloma and lenalidomide. Additionally, it is associated with a lower rate of peripheral neuropathy than bortezomib .
Alemtuzumab (Campath 1H), approved for the treatment of B-cell chronic lymphocytic leukemia, is a humanized monoclonal antibody binding to the CD52 antigen, highly expressed on the surface of B and T cells . The mechanism of action resulting in B-cell and T-cell depletion is not well understood. Depletion of T and B cells is observed soon after infusion, and recovery of these cells is gradual and takes about 6–12 months . Alemtuzumab has been used since 1998 in kidney transplantation as an induction agent and antirejection agent for both AMR and acute cellular rejection, because of its activity against both T and B cells . Interestingly, alemtuzumab also allows a low-dose CsA monotherapy. Only a few studies have reported the use of alemtuzumab for the treatment of AMR with or without cellular rejection [20–22]. In these studies, alemtuzumab was used with plasmapheresis and IVIg, with or without Rituximab. All of these studies reported an initial response to therapy and a decrease in serum creatinine within the first 2 days to 2 weeks of therapy. However, in one case of a fourth repeat kidney transplant a recurrence of AMR was reported 3 months posttherapy, as well as posttransplant lymphoproliferative disorder .
In another study, alemtuzumab was given to 101 children receiving living donor kidney transplantation, with the intention to eradicate peripheral lymphomonocytes and induce donor-specific tolerance. Two doses of 30 mg alemtuzumab were infused, one 12–29 days prior to transplantation and the other at surgery. The maintenance immunosuppression included low doses of calcineurin inhibitor drugs (CNIs) and mycophenolate mofetil (MMF). The minimum follow-up was 3 years. Graft survival was 96% at 1 year and 89% at 3 years. Acute rejection was detected with protocol biopsies in 26% of children at 1 year and in 35% at 2 years, while no rejection was detected thereafter . The main conclusion from this study was that alemtuzumab pretreatment before living donor kidney transplantation is a good option, allowing a reduction in the usual doses of CNI and achieving satisfactory middle-term results. A subsequent study investigated the effects of alemtuzumab, 0.5 mg/kg for a maximum of 30 mg, in 25 children undergoing cadaveric kidney transplantation. In this case the drug was given after anesthesia, but before kidney transplantation. 10 mg/kg of methyl prednisolone was given perioperatively and before revascularization. Children received steroid therapy for another 4 days and tacrolimus (TAC), as monotherapy, was initiated at day 1 . Recent randomized control trials showed that the use of alemtuzumab, compared with the use of basiliximab, led to a lower frequency of acute rejection in adult patients that were not at high immunological risk [25▪▪,26▪▪].
Beyond proteasome inhibition, anti-B-lymphocyte stimulating factor therapies – an example of which is the FDA-approved drug belimumab – are being used in autoantibody diseases and are being investigated for their potential use in solid organ transplantation. This group of compounds may also have the ability to kill the plasma cell and, therefore, may be effective in decreasing/removing DSA on its own or in combination with proteasome inhibitor therapy. Other therapies, such as tocilizumabs, an FDA-approved humanized monoclonal antibody against interleukin-6 receptor, or a more potent TOR inhibitor, such as temsirolimus, may also be useful in therapeutic regimens that are focused on antibody removal. More data on these novel compounds are still needed.
Eculizumab is a humanized anti-C5 monoclonal antibody that inhibits the formation of the C5–C9 complex or MAC. All cases reporting the use of Eculizumab in treating AMR were limited to highly sensitized patients with AMR refractory to other therapies [27–30]. AMR was resolved in 100% of the patients . Another new research product, able to interfere with humoral immunity activity, is C1 inhibitor concentrate. C1 blocking agents inhibit the first step of the complement cascade. Both medications are FDA approved for hereditary angioedema. C1 blockade was effective in preventing acute AMR in an animal study , but no clinical evidence yet exists in humans. In case of C1 inhibitors, one of the safety concerns, dose-related, is the occurrence of thrombotic events that were found in both animal and human studies . Trials to evaluate the efficacy and safety of C1 inhibitors in preventing or treating AMR in kidney transplant recipients are currently underway .
Some innovative therapies that aim to block the synthesis of DSA focus on the costimulatory signal. . Among these is an engineered soluble form of cytotoxic T-lymphocyte antigen 4 (CTLA-4) (CTLA4-Ig), belatacept, which binds to the surface proteins CD80 and CD86 found on antigen presenting cells, including B cells, and inhibits T-cell activation. Early clinical trials with belatacept showed promise as fewer patients developed DSA compared with cyclosporine-treated patients .
In pediatric kidney transplantation, belatacept is a promising agent for allowing steroid-free and CNI-free immunosuppression. In a recent report on a living donor kidney transplant [36▪▪], belatacept was used monthly in association with daily sirolimus. Following Alemtuzumab induction, belatacept and sirolimus prevented kidney allograft rejection without CNIs or steroids. The effect of a similar protocol in children is under investigation.
Another potential target for costimulation blockade is the B-cell costimulatory molecule CD40. Phase 2 clinical trials are in progress, examining the efficacy of an anti-CD40 antibody. Yet, other costimulatory inhibitors, for example an anti-CD28 antibody fragment, are being developed in order to prevent chronic humoral immune-mediated graft injury .
B cells require activation signals delivered by specific cytokines, which may prove to be effective targets in preventing DSA. One B-cell growth factor and B-cell-activating factor (BAFF) provides critical early signals for the development of B2 and marginal zone B cells . In human kidney transplantation, elevated BAFF or BAFF receptor levels correlated with rejection and graft dysfunction . However, a clinical trial of anti-BAFF therapy, which proved ineffective, was prematurely halted (http://clinicaltrials.gov/ct2/show/NCT01025193).
A specific B-cell maturation factor, APRIL (a proliferation-inducing ligand) favors the establishment/survival of late plasmablasts and promotes long-lived antibody responses. Clinical trials of APRIL-directed therapy have not yet been conducted.
The potential relevance of the B-cell-stimulating, growth and maturation promoting cytokine, interleukin 14 (IL14) in transplantation has also been evaluated . IL14 transcript levels in human T cells increase in vitro following allostimulation. In transplant recipients, IL14 mRNA levels are increased in those undergoing rejection or infection compared with stable patients . These data suggest that IL14 may prove to be a biomarker, indicating graft health, and may play a role in the generation of DSA after kidney transplantation. Further studies will be required to fully explore the role of B-cell-stimulating cytokines and the therapeutic benefits of targeting these factors.
In conclusion, AMR is common in patients with DSA and is associated with a poor prognosis. Novel medications that target each step of AMR pathogenesis have been produced and are successful when compared with more traditional therapies, as is shown by several (case) studies. Controlled studies are mandatory to confirm these positive results. Side-effects and cost are the only factors limiting the use of these medications in the treatment of AMR.
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Conflicts of interest
There are no conflicts of interest.
REFERENCES AND RECOMMENDED READING
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