To optimize strategies that mitigate the risk of graft loss associated with HLA incompatibility, we evaluated whether sequence defined HLA targets (eplets) that result in donor-specific antibodies ...are associated with transplant outcomes. To define this, we fit multivariable Cox proportional hazard models in a cohort of 118 382 United States first kidney transplant recipients to assess risk of death-censored graft failure by increments of ten antibody-verified eplet mismatches. To verify robustness of our findings, we conducted sensitivity analysis in this United States cohort and assessed the role of antibody-verified eplet mismatches as autonomous predictors of transplant glomerulopathy in an independent Canadian cohort. Antibody-verified eplet mismatches were found to be independent predictors of death-censored graft failure with hazard ratios of 1.231 95% confidence interval 1.195, 1. 268, 1.268 1.231, 1.305 and 1.411 1.331, 1.495 for Class I (HLA-A, B, and C), -DRB1 and -DQB1 loci, respectively. To address linkage disequilibrium between HLA-DRB1 and -DQB1, we fit models in a subcohort without HLA-DQB1 eplet mismatches and found hazard ratios for death-censored graft failure of 1.384 1.293, 1.480 for each additional antibody-verified HLA-DRB1 eplet mismatch. In a subcohort without HLA-DRB1 mismatches, the hazard ratio was 1.384 1.072, 1.791 for each additional HLA-DQB1 mismatch. In the Canadian cohort, antibody-verified eplet mismatches were independent predictors of transplant glomerulopathy with hazard ratios of 5.511 1.442, 21.080 for HLA-DRB1 and 3.640 1.574, 8.416 for -DRB1/3/4/5. Thus, donor-recipient matching for specific HLA eplets appears to be a feasible and clinically justifiable strategy to mitigate risk of graft loss.
Display omitted
Abstract The International Registry of Antibody-Defined HLA Epitopes ( http://www.epregistry.com.br ) has been recently established as a tool to understand humoral responses to HLA mismatches. These ...epitopes can be structurally defined as eplets by three-dimensional molecular modeling and amino acid sequence differences between HLA antigens. A major goal is to identify HLA eplets that have been verified experimentally with informative antibodies. This report addresses class II epitopes encoded by genes in the HLA-D region. Our analysis included reviews of many publications about epitope specificity of class II reactive human and murine monoclonal antibodies and informative alloantibodies from HLA sensitized patients as well as our own antibody testing results. As of July 1, 2014, 24 HLA-DRB1/3/4/5, 15 DQB, 3 DQA and 8 DPB antibody-verified epitopes have been identified and recorded. The Registry is still a work-in-progress and will become a useful resource for HLA professionals interested in histocompatibility testing at the epitope level and investigating antibody responses to HLA mismatches in transplant patients.
Abstract Serum analysis of patients considered for retransplantation has a potential limitation that the rejected allograft may absorb HLA antibodies. We have determined how the highly sensitive ...micro bead-based Luminex antibody-binding assay with single antigens can detect donor-specific HLA antibodies (DSA) in patients before and after surgical removal of a rejected allograft. This analysis was done for 65 allograft nephrectomy (allonx) cases contributed by 16 laboratories worldwide. In the HLA-A,B and -DRB1 mismatch categories the incidence of DSA reactivity pre-allonx and post-allonx was 64% vs 87% ( p = 0.0033) and 57% vs 86% ( p = 0.001), respectively. The frequencies of individual reactive antigens were also lower before allonx: for HLA-A,B antigens: 49% vs 75% ( p < 0.0001) and DRB1 antigens: 48% vs 79% ( p = 0.0001). On the other hand, no significant differences were seen between the pre-allonx and post-allonx frequencies of DSA to DRB3/4/5 (65% vs 78%, p = 0.22) and DQ mismatches (76% vs 87%, p = 0.18). Conclusion: although the sensitive Luminex antibody assay can detect anti-donor antibodies in the presence of a rejected transplant, it is apparent that the antibody specificity pattern is often incomplete especially against the HLA-A, -B and DR mismatches. This understanding seems relevant to the determination of acceptable mismatches for patients considered for retransplantation.
As methods for human leukocyte antigens (HLA) antibody detection have evolved and newer solid phase assays are much more sensitive, the last 15 years has seen a renewed focus on the importance of HLA ...antibodies in solid organ transplant rejection. However, there is still much controversy regarding the clinical significance of antibody level as depicted by the mean fluorescence intensity of a patient's neat serum. Emerging techniques, including those that identify antibody level and function, show promise for the detection of individuals at risk of allograft rejection, determination of the effectiveness of desensitization prior to transplant, and for monitoring treatment of rejection. Here, we review current publications regarding the relevance of donor-specific HLA antibodies (DSA) in adult and pediatric heart transplantation (HT) with graft survival, development of antibody-mediated rejection and cardiac allograft vasculopathy (CAV). The negative impact of DSA on patient and allograft survival is evident in adult and pediatric HT recipients. Many questions remain regarding the most appropriate frequency of assessment of pre- and posttransplant DSA as well as the phenotype of DSA memory vs. true
antibody using large multicenter adult and pediatric cohorts and state-of-the-art methodologies for DSA detection and characterization.
Abstract HLA matching at the epitope level offers new opportunities to identify suitable donors for transplant patients. The International HLA Epitope Registry ( www.Epregistry.com.br ) describes for ...the various HLA loci, repertoires of eplets including those that correspond to epitopes experimentally verified with specific antibodies. There are also many eplets which have remained as theoretical entities because no informative antibodies have been found. Which of them have immunogenic potential or conversely, might be considered as non-epitopes that cannot elicit specific antibody responses? This question is important for the application of epitope-based HLA matching in clinical transplantation. Correct predictions of B-cell epitopes on antigenic proteins are essential to the effective design of microbial vaccines and the development of specific antibodies used in immunotherapy and immunodiagnostics but prediction programs based on structural and physiochemical properties of amino acid residues are generally ineffective. Recent prediction programs based on three-dimensional structures of antigen-antibody complexes are more promising. One such program is called ElliPro developed by Ponomarenko. This report describes studies demonstrating that ElliPro can predict alloantibody responses to HLA-ABC eplets. Antibody-verified eplets have amino acid residues with much higher ElliPro scores than eplets for which no specific antibodies have been found. The latter group includes residues with very low ElliPro scores; they appear to represent eplets that might be classified as non-epitopes. In conclusion, ElliPro offers a new approach to characterize epitope repertoires that are clinically relevant in HLA matching.
Although antibody-mediated rejection (ABMR) has been long recognized as a leading cause of allograft failure after kidney transplantation, the cellular and molecular processes underlying the ...induction of deleterious donor-specific antibody (DSA) responses remain poorly understood.
Using high-dimensional flow cytometry,
assays, and RNA sequencing, we concomitantly investigated the role of T follicular helper (T
) cells and B cells during ABMR in 105 kidney transplant recipients.
There were 54 patients without DSAs; of those with DSAs, ABMR emerged in 20 patients, but not in 31 patients. We identified proliferating populations of circulating T
cells and activated B cells emerging in blood of patients undergoing ABMR. Although these circulating T
cells comprised heterogeneous phenotypes, they were dominated by activated (ICOS
PD-1
) and early memory precursor (CCR7
CD127
) subsets, and were enriched for the transcription factors IRF4 and c-Maf. These circulating T
cells produced large amounts of IL-21 upon stimulation with donor antigen and induced B cells to differentiate into antibody-secreting cells that produced DSAs. Combined analysis of the matched circulating T
cell and activated B cell RNA-sequencing profiles identified highly coordinated transcriptional programs in circulating T
cells and B cells among patients with ABMR, which markedly differed from those of patients who did not develop DSAs or ABMR. The timing of expansion of the distinctive circulating T
cells and activated B cells paralleled emergence of DSAs in blood, and their magnitude was predictive of IgG3 DSA generation, more severe allograft injury, and higher rate of allograft loss.
Patients undergoing ABMR may benefit from monitoring and therapeutic targeting of T
cell-B cell interactions.
The presence of antibodies to angiotensin type 1 receptor (AT1R) and endothelin type A receptor (ETAR) is associated with allograft rejection in kidney and heart transplantation. The aim of our study ...was to determine the impact of AT1R and ETAR antibodies on graft outcome in lung transplantation.
Pretransplant and posttransplant sera from 162 lung recipients transplanted at 3 centers between 2011 and 2013 were tested for antibodies to AT1R and ETAR by the enzyme-linked immunosorbent assay (ELISA) assay. Clinical parameters analyzed were: HLA antibodies at transplant, de novo donor-specific antibodies (DSA), antibody-mediated rejection (AMR), acute cellular rejection, and graft status.
Late AMR (median posttransplant day 323) was diagnosed in 5 of 36 recipients with de novo DSA. Freedom from AMR significantly decreased for those recipients with strong/intermediate binding antibodies to AT1R (P = 0.014) and ETAR (P = 0.005). Trends for lower freedom from acute cellular rejection were observed for recipients with pretransplant antibodies to AT1R (P = 0.19) and ETAR (P = 0.32), but did not reach statistical significance. Lower freedom from the development of de novo DSA was observed for recipients with antibodies detected pretransplant to AT1R (P = 0.054), ETAR (P = 0.012), and HLA-specific antibodies (P = 0.063). When the pretransplant antibody status of HLA-specific antibody (hazard ratio HR, 1.69) was considered together with either strong binding to AT1R or ETAR, an increased negative impact on the freedom from the development of de novo DSA was observed (HR, 2.26 for HLA antibodies and ETAR; HR, 2.38 for HLA antibodies and ETAR).
These results illustrate the increased negative impact when antibodies to both HLA and non-HLA antigens are present pretransplant.
Human immunoglobulins (H-Ig) are widely used in solid organ transplantation for immunoglobulin G (IgG) replacement and for desensitization and treatment of antibody-mediated rejection. They are ...obtained from plasma pools and may contain HLA antibodies that can be detrimental to transplant recipients. The goal of this study was to evaluate HLA antibodies in multiple lots of 2 commercial H-Ig preparations by Luminex single-antigen bead (SAB) and cell-based crossmatch assays.
Thirty lots of 2 commercial H-Ig products (CSL Behring, King of Prussia, PA) were evaluated: 6 Hizentra and 24 Privigen. All were adsorbed and diluted 1:10 before testing. HLA IgG antibodies were determined by 2 Luminex SAB kits and C1q screen for complement-binding capability. Lots were tested for the presence of antibody to denatured vs. intact class I HLA alleles using acid-treated SAB. Surrogate T and B-cell flow cytometry crossmatches (FCXM) were performed with peripheral blood lymphocytes from 2 healthy donors.
Twenty-two (73%) lots at 1:10 showed SAB reactivity with mean fluorescent intensity of 2000 or greater for HLA class I, 67% (20/30 lots) for class II. The reactivity pattern was similar using both SAB kits. Acid treatment revealed antibodies to denatured class I: the majority of HLA-C, half of HLA-B and few HLA-A alleles. No C1q reactivity was observed. Surrogate flow cytometry crossmatch results were positive (>150 median channel shift), but were fourfold to eightfold lower than expected.
The H-Ig products tested consisted of low titer, non-complement-binding HLA class I and class II antibodies; most of the observed class I HLA reactivity was toward denatured HLA antigens.
The presence of HLA antibodies is widely recognized as a barrier to solid organ transplantation, and for lung transplant candidates, it has a significant negative impact on both waiting time and ...waiting list mortality. Although HLA antibodies have been associated with a broad spectrum of allograft damage, precise characterization of these antibodies in allosensitized candidates may enhance their accessibility to transplant. The introduction of Luminex-based single antigen bead (SAB) assays has significantly improved antibody detection sensitivity and specificity, but SAB alone is not sufficient for risk-stratification. Functional characterization of donor-specific antibodies (DSA) is paramount to increase donor accessibility for allosensitized lung candidates. We describe here our approach to evaluate sensitized lung transplant candidates. By employing state-of-the-art technologies to assess histocompatibility and determine physiological properties of circulating HLA antibodies, we can provide our Clinical Team a better risk assessment for lung transplant candidates and facilitate a "road map" to transplant. The cases presented in this paper illustrate the "individualized steps" taken to determine calculated panel reactive antibodies (cPRA), titer and complement-fixing properties of each HLA antibody present in circulation. When a donor is considered, we can better predict the risk associated with potentially crossing HLA antibodies, thereby allowing the Clinical Team to approach allosensitized lung patients with an individualized medicine approach. To facilitate safe access of sensitized lung transplant candidates to potential donors, a synergy between the histocompatibility laboratory and the Clinical Team is essential. Ultimately, donor acceptance is a decision based on several parameters, leading to a risk-stratification unique for each patient.
PDF
