Elaine F. Reed

Consensus guidelines on the testing and clinical management issues associated with HLA and non-HLA antibodies in transplantation.

Background The introduction of solid-phase immunoassay (SPI) technology for the detection and characterization of human leukocyte antigen (HLA) antibodies in transplantation while providing greater sensitivity than was obtainable by complement-dependent lymphocytotoxicity (CDC) assays has resulted in a new paradigm with respect to the interpretation of donor-specific antibodies (DSA). Although the SPI assay performed on the Luminex instrument (hereafter referred to as the Luminex assay), in particular, has permitted the detection of antibodies not detectable by CDC, the clinical significance of these antibodies is incompletely understood. Nevertheless, the detection of these antibodies has led to changes in the clinical management of sensitized patients. In addition, SPI testing raises technical issues that require resolution and careful consideration when interpreting antibody results. Methods With this background, The Transplantation Society convened a group of laboratory and clinical experts in the field of transplantation to prepare a consensus report and make recommendations on the use of this new technology based on both published evidence and expert opinion. Three working groups were formed to address (a) the technical issues with respect to the use of this technology, (b) the interpretation of pretransplantation antibody testing in the context of various clinical settings and organ transplant types (kidney, heart, lung, liver, pancreas, intestinal, and islet cells), and (c) the application of antibody testing in the posttransplantation setting. The three groups were established in November 2011 and convened for a “Consensus Conference on Antibodies in Transplantation” in Rome, Italy, in May 2012. The deliberations of the three groups meeting independently and then together are the bases for this report. Results A comprehensive list of recommendations was prepared by each group. A summary of the key recommendations follows. Technical Group: (a) SPI must be used for the detection of pretransplantation HLA antibodies in solid organ transplant recipients and, in particular, the use of the single-antigen bead assay to detect antibodies to HLA loci, such as Cw, DQA, DPA, and DPB, which are not readily detected by other methods. (b) The use of SPI for antibody detection should be supplemented with cell-based assays to examine the correlations between the two types of assays and to establish the likelihood of a positive crossmatch (XM). (c) There must be an awareness of the technical factors that can influence the results and their clinical interpretation when using the Luminex bead technology, such as variation in antigen density and the presence of denatured antigen on the beads. Pretransplantation Group: (a) Risk categories should be established based on the antibody and the XM results obtained. (b) DSA detected by CDC and a positive XM should be avoided due to their strong association with antibody-mediated rejection and graft loss. (c) A renal transplantation can be performed in the absence of a prospective XM if single-antigen bead screening for antibodies to all class I and II HLA loci is negative. This decision, however, needs to be taken in agreement with local clinical programs and the relevant regulatory bodies. (d) The presence of DSA HLA antibodies should be avoided in heart and lung transplantation and considered a risk factor for liver, intestinal, and islet cell transplantation. Posttransplantation Group: (a) High-risk patients (i.e., desensitized or DSA positive/XM negative) should be monitored by measurement of DSA and protocol biopsies in the first 3 months after transplantation. (b) Intermediate-risk patients (history of DSA but currently negative) should be monitored for DSA within the first month. If DSA is present, a biopsy should be performed. (c) Low-risk patients (nonsensitized first transplantation) should be screened for DSA at least once 3 to 12 months after transplantation. If DSA is detected, a biopsy should be performed. In all three categories, the recommendations for subsequent treatment are based on the biopsy results. Conclusions A comprehensive list of recommendations is provided covering the technical and pretransplantation and posttransplantation monitoring of HLA antibodies in solid organ transplantation. The recommendations are intended to provide state-of-the-art guidance in the use and clinical application of recently developed methods for HLA antibody detection when used in conjunction with traditional methods.

Assessing relative risks of infection and rejection : A meta-analysis using an immune function assay

Background. Long-term use of immunosuppressants is associated with significant morbidity and mortality in transplant recipients. A simple whole blood assay that has U.S. Food and Drug Administration clearance directly assesses the net state of immune function of allograft recipients for better individualization of therapy. A meta-analysis of 504 solid organ transplant recipients (heart, kidney, kidney-pancreas, liver and small bowel) from 10 U.S. centers was performed using the Cylex ImmuKnow assay. Methods. Blood samples were taken from recipients at various times posttransplant and compared with clinical course (stable, rejection, infection). In this analysis, 39 biopsy-proven cellular rejections and 66 diagnosed infections occurred. Odds ratios of infection or rejection were calculated based on measured immune response values. Results. A recipient with an immune response value of 25 ng/ml adenosine triphosphate (ATP) was 12 times (95% confidence of 4 to 36) more likely to develop an infection than a recipient with a stronger immune response. Similarly, a recipient with an immune response of 700 ng/ml ATP was 30 times (95% confidence of 8 to 112) more likely to develop a cellular rejection than a recipient with a lower immune response value. Of note is the intersection of odds ratio curves for infection and rejection in the moderate immune response zone (280 ng/ml ATP). This intersection of risk curves provides an immunological target of immune function for solid organ recipients. Conclusion. These data show that the Cylex ImmuKnow assay has a high negative predictive value and provides a target immunological response zone for minimizing risk and managing patients to stability.

Report from a consensus conference on antibody-mediated rejection in heart transplantation

BACKGROUND The problem of AMR remains unsolved because standardized schemes for diagnosis and treatment remains contentious. Therefore, a consensus conference was organized to discuss the current status of antibody-mediated rejection (AMR) in heart transplantation. METHODS The conference included 83 participants (transplant cardiologists, surgeons, immunologists and pathologists) representing 67 heart transplant centers from North America, Europe, and Asia who all participated in smaller break-out sessions to discuss the various topics of AMR and attempt to achieve consensus. RESULTS A tentative pathology diagnosis of AMR was established, however, the pathologist felt that further discussion was needed prior to a formal recommendation for AMR diagnosis. One of the most important outcomes of this conference was that a clinical definition for AMR (cardiac dysfunction and/or circulating donor-specific antibody) was no longer believed to be required due to recent publications demonstrating that asymptomatic (no cardiac dysfunction) biopsy-proven AMR is associated with subsequent greater mortality and greater development of cardiac allograft vasculopathy. It was also noted that donor-specific antibody is not always detected during AMR episodes as the antibody may be adhered to the donor heart. Finally, recommendations were made for the timing for specific staining of endomyocardial biopsy specimens and the frequency by which circulating antibodies should be assessed. Recommendations for management and future clinical trials were also provided. CONCLUSIONS The AMR Consensus Conference brought together clinicians, pathologists and immunologists to further the understanding of AMR. Progress was made toward a pathology AMR grading scale and consensus was accomplished regarding several clinical issues.

The role of anti-HLA antibodies in heart transplantation

The major threat to long-term survival of heart allograft recipients is the development of graft atherosclerosis, which seems to be a manifestation of chronic rejection. To assess the role of anti-HLA antibodies in heart allograft rejection we studied 107 patients and compared the survival of recipients who formed anti-HLA antibodies with the survival of recipients who developed no antibodies. At 4 years the actuarial survival was 90% in the nonproducer group and 38% in antibody-producers (P = 0.038). We further explored the possibility that HLA antigens from the injured graft are released into the circulation and can be found in the serum either free or complexed with anti-HLA antibodies. This hypothesis was confirmed by the finding that the frequency of sera containing soluble HLA antigens from the graft or immune complexes of HLA alloantigens with anti-HLA antibodies was significantly higher in patients who rejected compared with patients with successful heart allografts (P less than 0.05). Following depletion of soluble HLA antigens, anti-HLA antibodies became detectable in 53% and 74% sera obtained during the first and second year posttransplantation, respectively, from patients undergoing chronic rejection. Long-term survivors showed a significantly lower (P less than 0.001) frequency of anti-HLA antibodies in sera depleted of HLA antigens. Lastly, studies of anti-anti-HLA-A2 and A3 antibodies in recipient sera suggest that quiescence is maintained by antiidiotypic antibodies.

Asymptomatic Antibody-mediated Rejection After Heart Transplantation Predicts Poor Outcomes

BACKGROUND Antibody-mediated rejection (AMR) has been associated with poor outcome after heart transplantation. The diagnosis of AMR usually includes endomyocardial biopsy findings of endothelial cell swelling, intravascular macrophages, C4d+ staining, and associated left ventricular dysfunction. The significance of AMR findings in biopsy specimens of asymptomatic heart transplant patients (normal cardiac function and no symptoms of heart failure) is unclear. METHODS Between July 1997 and September 2001, AMR was found in the biopsy specimens of 43 patients. Patients were divided into 2 groups: asymptomatic AMR (AsAMR, n = 21) and treated AMR (TxAMR with associated left ventricular dysfunction, n = 22). For comparison, a control group of 86 contemporaneous patients, without AMR, was matched for age, gender, and time from transplant. Outcomes included 5-year actuarial survival and development of cardiac allograft vasculopathy (CAV). Patients were considered to have AMR if they had > or = 1 endomyocardial biopsy specimen positive for AMR. RESULTS The 5-year actuarial survival for the AsAMR (86%), TxAMR (68%), and control groups (79%) was not significantly different (p = 0.41). Five-year freedom from CAV (> or = 30% stenosis in any vessel) was AsAMR, 52%; TxAMR, 68%; and control, 79%. Individually, freedom from CAV was significantly lower in the AsAMR group compared with the control group (p = 0.02). There was no significant difference between AsAMR vs TxAMR and TxAMR vs control for CAV. CONCLUSIONS Despite comparable 5-year survival with controls after heart transplantation, AsAMR rejection is associated with a greater risk of CAV. Trials to treat AsAMR to alter outcome are warranted.

Effect of Antibodies on Endothelium

Patients developing posttransplant antibodies against HLA and non‐HLA antigens expressed by the endothelium of the graft undergo more frequent episodes of rejection and have decreased long‐term graft survival. Antibodies against the endothelium can alter/damage the cells of the graft through several mechanisms. Historically, antibodies were thought to elicit endothelial cell injury via complement‐dependent mechanisms. New research has shown that antibodies can also contribute to the process of transplant rejection by stimulating proinflammatory and proproliferation signals. Antibody ligation leads to several functional alterations in EC including Weibel Palade body exocytosis, leukocyte recruitment, growth factor expression and cell proliferation. In contrast, under certain circumstances, antibodies may induce prosurvival signals and graft accommodation. The signaling events regulating accommodation vs. rejection appear to be influenced by the specificity and concentration of the anti‐HLA antibody and the degree of molecular aggregation. Knowledge of the HLA and non‐HLA antibody‐mediated signaling pathways has the potential to identify new therapeutic targets to promote accommodation and prevent acute and chronic antibody‐mediated rejection.

Monitoring of soluble HLA alloantigens and anti-HLA antibodies identifies heart allograft recipients at risk of transplant-associated coronary artery disease.

The development of accelerated transplant-related coronary artery disease (T-CAD) is the major obstacle to long-term survival of cardiac allografts. We have investigated the role of various demographic and immunologic parameters as prognostic indicators of T-CAD in a population of 274 heart allograft recipients. Our data demonstrate that patients who experience more than 1 episode of acute rejection per year and/or develop antidonor HLA antibodies are at increased risk of developing T-CAD. Using HLA-A2 as a marker for the release of soluble HLA antigens from the donor, we established that recipients displaying circulating donor alloantigens for more than 26 weeks following transplantation are at increased risk of developing T-CAD (P=0.008). This association suggests that the release of alloantigens from the allograft is indicative of chronic injury and/or that it stimulates chronic rejection via the indirect allorecognition pathway. Our findings indicate that patients at risk of developing T-CAD can be identified by monitoring the release of donor alloantigens and production of antidonor HLA antibodies following transplantation.

Effect of Antiidiotypic Antibodies to HLA on Graft Survival in Renal-Allograft Recipients

Although the presence in the recipient of preformed antibodies to HLA antigens in the kidney of a renal-transplant donor may be associated with early graft failure, such grafts are often well tolerated. We have investigated the possibility that anti-anti-HLA (antiidiotypic) antibodies influence the outcome of renal transplantation in recipients with a history of presensitization to their donors HLA antigens. A retrospective analysis of 20 such cases showed that in 10 patients the transplanted kidney was rejected within one month, whereas in the remaining 10 the graft was tolerated for more than a year. Nine of the 10 patients in whom the graft was tolerated had anti-anti-HLA antibodies at the time of transplantation. Nine of the 10 patients in whom the graft was rejected had antibodies that potentiated, rather than blocked, the cytotoxic activity of anti-donor-HLA antibodies. These results suggest that patients with anti-anti-HLA antibodies specific for a potential donor can safely undergo transplantation, despite a prior history of anti-HLA antibodies. At the time of transplantation, patients who have antibodies that potentiate the cytotoxic activity of a historically positive serum are at high risk of graft rejection within a short period. Taking these considerations into account may improve the reliability of cross-matching in renal transplantation.

The Banff 2015 Kidney Meeting Report: Current Challenges in Rejection Classification and Prospects for Adopting Molecular Pathology

The XIII Banff meeting, held in conjunction the Canadian Society of Transplantation in Vancouver, Canada, reviewed the clinical impact of updates of C4d‐negative antibody‐mediated rejection (ABMR) from the 2013 meeting, reports from active Banff Working Groups, the relationships of donor‐specific antibody tests (anti‐HLA and non‐HLA) with transplant histopathology, and questions of molecular transplant diagnostics. The use of transcriptome gene sets, their resultant diagnostic classifiers, or common key genes to supplement the diagnosis and classification of rejection requires further consensus agreement and validation in biopsies. Newly introduced concepts include the i‐IFTA score, comprising inflammation within areas of fibrosis and atrophy and acceptance of transplant arteriolopathy within the descriptions of chronic active T cell–mediated rejection (TCMR) or chronic ABMR. The pattern of mixed TCMR and ABMR was increasingly recognized. This report also includes improved definitions of TCMR and ABMR in pancreas transplants with specification of vascular lesions and prospects for defining a vascularized composite allograft rejection classification. The goal of the Banff process is ongoing integration of advances in histologic, serologic, and molecular diagnostic techniques to produce a consensus‐based reporting system that offers precise composite scores, accurate routine diagnostics, and applicability to next‐generation clinical trials.

HLA Class I Antibody-Mediated Endothelial Cell Proliferation via the mTOR Pathway

Anti-HLA Abs have been shown to contribute to the process of transplant vasculopathy by binding to HLA class I molecules expressed by the endothelial and smooth muscle cells of the graft and transducing intracellular signals that elicit cell proliferation. The aim of this study was to determine the role of mammalian target of rapamycin (mTOR) in HLA class I-induced endothelial cell proliferation and to explore in depth the relationship between mTOR complexes and their downstream targets following ligation of HLA class I molecules by anti-HLA Abs. We used small interfering RNA technology to abrogate mTOR, rapamycin-insensitive companion of mTOR (rictor), or regulatory associated protein of mTOR (raptor) to study the function of these gene products to activate proteins involved in MHC class I-induced cell proliferation and survival. Knockdown of mTOR inhibited class I-mediated phosphorylation of proteins downstream of mTOR complex 1 and mTOR complex 2. Furthermore, knockdown of mTOR, rictor, or raptor blocked HLA class I-induced endothelial cell proliferation. Long-term pretreatment with the mTOR inhibitor rapamycin significantly blocked both mTOR-raptor and mTOR-rictor complex formation. Interestingly, rapamycin also blocked class I-induced Akt phosphorylation at Ser473 and Bcl-2 expression. These results support the role of anti-HLA Abs in the process of transplant vasculopathy and suggest that exposure of the graft endothelium to anti-HLA Abs may promote proliferation through the mTOR pathway.