The single antigen expressing lines (SALs) concept: an excellent tool for screening for HLA-specific antibodies

Definition of the antibody specificity in the serum of patients waiting for a renal transplant or in need for platelet transfusion is a crucial step for finding adequate donors. Confounding factors are the complexity of the serum antibodies and the expression of several, up to six, different human leukocyte antigens (HLA) on peripheral blood lymphocytes used as target cells in the antibody screening. Single antigen-expressing (SAL) cell lines were generated by transfecting human major histocompatibility complex (MHC) class I sequences into K562, an erythroleukemia-derived cell line lacking MHC class I and II expression. Thirty-seven different SALs have been generated so far. In this study, we present the validation of 16 of those SALs by flow cytometry against a panel of 84 human HLA-specific monoclonal antibodies (30 HLA-A [8 IgG/22 IgM], 45 HLA-B [18 IgG/27 IgM], 6 HLA-A, B [3 IgG/3 IgM], and 3 HLA-C [all IgM]) developed in our laboratory. The SALs proved to be suitable tools to determine acceptable mismatches for highly sensitized patients. This concept of transfecting target sequences in immortalized cell lines opens up new avenues in the definition of serum and cellular reactivity for sensitized patients awaiting a suitable organ or blood component.     

A limited number of HLA epitopes are recognized from HLA class I-specific antibodies detected in the serum of sensitized patients

The goal of this study was to evaluate the epitope specificity of HLA class I-specific antibodies detected in the serum of sensitized patients awaiting retransplantation. The study group consisted of 22 sensitized from previous graft patients, who produced stable IgG HLA class I-specific antibodies. A total of 60 serum samples were screened and analyzed by two techniques in parallel: the antihuman globulin augmented CDC (AHG-CDC) technique and an ELISA technique. All recipients and donors were typed for class I HLA antigens by a standard lymphocytotoxicity technique. The epitope identification was based on class I HLA antigens sequencing, where the multiple immunogenic epitopes are differentially shared among various HLA antigens. The unique epitope configuration on one HLA antigen represents the private epitope of the specific HLA antigen while epitopes shared by more than one HLA antigen represent public determinants. In some HLA antigens (HLA-A1), more than one private epitope has been defined, while in others (HLA-B35, -B51), the private epitopes are not yet known. In a total of 36 antibody reactivity patterns, the majority of the definable IgG HLA class I-specific antibodies corresponded to the A-locus (75%), and only 25% had specificities against the B-locus antigens, although the number of incompatibilities concerning both loci were almost identical (29 for the HLA antigens of the A-locus and 26 for those of B-locus). All patients produced HLA class I-specific antibodies with specificities against the private epitopes of the immunogenic mismatched HLA antigen(s). In 6/21 cases (28.6%), HLA class I alloreactivity spreading to nongraft HLA antigens was detected and 9 public (shared) immunogenic alloepitopes were recognized. In conclusion, appling the epitope analysis of HLA class I-specific antibodies produced by sensitized from previous graft patients, we were able to define the immunogenic alloepitopes. We consider that the immunogenic alloepitopes, during transplantation course, are mainly private epitopes of mismatched HLA antigens and, in certain cases, shared epitopes between the donor alloantigens and other HLA antigens. This knowledge may offer the potential of transplanting sensitized patients through improved donor selection.     

First report on the antibody verification of MICA epitopes recorded in the HLA epitope registry

The International Registry of HLA Epitopes ( http://epregistry.com.br ) has been recently established as a tool to understand antibody responses to HLA mismatches. These epitopes are defined structurally by three‐dimensional molecular modelling and amino acid sequence differences between HLA antigens. A major goal was to identify HLA epitopes that have been verified experimentally with informative antibodies. This report addresses the identification of MICA epitopes. Our analysis included published information about MICA antibody reactivity in sera from sensitized patients as well as data from our own laboratories. This report describes twenty‐one MICA epitopes verified with antibodies which have primarily been tested in Luminex assays with single alleles. The epitopes correspond to distinct eplets that are often defined by single residues. 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.     

HLAMatchmaker: a molecularly based algorithm for histocompatibility determination. II. Verification of the algorithm and determination of the relative immunogenicity of amino acid triplet-defined epitopes

HLAMatchmaker is a computer algorithm that assesses human leukocyte antigen (HLA) compatibility at the structural level by intralocus and interlocus comparisons of polymorphic amino acid triplets in antibody-accessible sequences of HLA class I molecules. This program permits the identification of mismatched HLA antigens that share all of their polymorphic triplets with the patient's HLA antigens and, therefore, could be considered fully compatible. The validity of this algorithm has been verified by analyzing the antibody specificity patterns of 127 well-characterized sera (panel reactive antibody [PRA] > 80%) that had been screened by direct complement-dependent and/or anti-human globulin augmented lymphocytotoxicity testing with large HLA-typed cell panels. A 2 x 2 table-based Chi-square analysis program was applied to determine positive and negative correlations between serum reactivity and the presence HLA triplets assigned from the HLA types in the cell panel. The results indicate that high PRA patients do not produce antibodies to shared triplets on mismatched HLA antigens. Moreover, this serum analysis has permitted the identification of triplets with different degrees of immunogenicity as indicated by the frequencies of positive and negative correlations of serum reactivity with the HLA-typed cell panel. Mismatching for triplets with low immunogenicity provides further opportunities for identifying donors with acceptable HLA mismatches for highly sensitized patients.     

Should epitope-based HLA compatibility be used in the kidney allocation system?

The new kidney allocation system (KAS) still applies donor-recipient HLA compatibility mostly at the antigen level and although some four-digit alleles have been included. This system is used to record unacceptable mismatches for sensitized transplant candidates with serum HLA antibodies. Since the reactivities of such antibodies are specifically associated with epitopes rather than HLA antigens, a more scientifically accurate assessment of mismatch acceptability could be based on epitopes. HLA class I and class II epitope specificity analyses can now be readily performed with serum antibody assays with single allele panels. This report describes an epitope-based HLA compatibility system for KAS and involves recipient and donor HLA typing at the four-digit allele level. It focuses on sensitized patients who have serum antibodies specific for HLA epitopes that can be entered as unacceptable mismatches in the transplant candidate database. Newly developed software programs could readily identify compatible HLA types.     

Challenging the golden standard in defining donor-specific antibodies: does the solid phase assay meet the expectations?

The purpose of the study was to compare three different methods defining donor-specific antibodies (DSA): complement-dependent cytotoxicity (CDC), the flow cytometry method (FCM), and a special for that purpose commercially available Luminex-based solid phase assay (SPA). A panel of human monoclonal antibodies (HuMabs) with well-defined human leukocyte antigen (HLA) specificities was used as antibody source and single HLA antigen expressing cell lines (SAL) were used as targets. Two methods yielded identical results (CDC and FCM). However, the SPA, the method by which solubilized HLA molecules from the SAL are captured by microspheres, showed two additional reactions which could not be explained, neither by the epitope recognized by the HuMab nor by the widely accepted sensitivity of the SPA methodology. These unexplained results suggest that by capturing solubilized HLA molecules on microspheres, conformational changes might occur. Positive results obtained by similar Luminex-based microsphere methods should be therefore taken with caution and the 'recognized' HLA antigens should not automatically be considered as unacceptable for transplantation.     

HLA compatibility assessment and management of highly sensitized patients under the new kidney allocation system (KAS): A 2016 status report from twelve HLA laboratories across the U.S.

Twelve HLA laboratories were surveyed to assess the methods and operational issues involved to define highly sensitized patients and to assess HLA compatibility under the new kidney allocation system (KAS) in the U.S. All laboratories used single antigen bead assays both pre- and post-KAS to define both broad and allele-specific HLA antibodies. The methods and threshold used to list HLA unacceptable antigens in UNet for virtual crossmatch (vXM) and the criteria used for determining HLA compatibility varied among laboratories. Laboratories reported several limitations of the current assays including the accuracy of quantifiable antibody fluorescence values, inadequate coverage of common alleles on the bead panels, and challenges in calibrating the vXM. The new KAS has resulted in a significant surge of deceased donor organ offers requiring vXM evaluation under tight time constraints. In the post-KAS period, eight of twelve laboratories (67%) indicated that their center did not proceed to transplant based on vXM without a prospective lymphocyte crossmatch. In conclusion, HLA laboratories play a critical role in deceased donor allocation for highly sensitized patients under the new KAS. Significant opportunities exist to improve the methods used in the assessment of HLA compatibility to safely transplant highly sensitized patients.     

The antibody response to an HLA mismatch: a model for nonself-self discrimination in relation to HLA epitope immunogenicity

Antibodies to HLA mismatches are specific for epitopes rather than antigens. HLAMatchmaker considers each HLA antigen as a string of eplets that represent key elements of epitopes. Certain antibodies are specific for single eplets, but recent studies have demonstrated that epitopes defined by eplet pairs always involve one nonself‐eplet and a self‐eplet shared between the immunizing antigen and the antibody producer. This suggests an autoreactive component of the alloantibody response to an HLA mismatch and this report expands this concept. During B‐cell development, V_H and V_L gene rearrangements produce a diversity of Ig receptors that can recognize epitopes on autologous proteins. It is hypothesized that B cells carry low‐affinity receptors for self‐HLA antigens. Their interactions with self‐HLA proteins will not lead to B‐cell activation or antibody production. In contrast, exposure to HLA mismatches induces often strong alloantibody responses. The activation of self‐HLA‐specific B cell by a nonself‐eplet may require that the remainder of the structural epitope of the immunizing antigen has considerable structural similarity with one of the antibody producer’s alleles. This hypothesis has been tested in molecular modelling studies with six epitopes defined by human monoclonal antibodies. In each case, one allele of the antibody producer had no or few differences with the immunizing allele in antibody‐accessible positions defined by a 15 Ångstrom radius of the mismatched eplet. The other alleles of the antibody producer showed significantly greater numbers of residue differences with the immunizer (5.8 ± 2.0 versus 1.0 ± 0.6, P < 0.0001). These data support the concept that HLA antibodies originate from B cells with self‐HLA immunoglobulin receptors that recognize mismatched eplets as nonself entities on immunizing antigens. The nonself–self paradigm provides a new insight of HLA epitope immunogenicity and may explain why sensitized patients have antibodies to a restricted number of mismatched epitopes.     

First report on the antibody verification of HLA-DR,HLA-DQ and HLA-DP epitopes recorded in the HLA Epitope Registry

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.     

HLA epitopes – Empirically defined as conformational amino acids sequences of the HLA antigen and are likely to be part of the binding sites of anti-HLA antibodies

Antibodies against HLA antigens are ubiquitous in the sera of transplant patients. Analysis of anti-HLA antibodies specificity has gone through a long history of development using assays like agglutination and lymphocytotoxicity, which utilize lymphocytes, and flow cytometry, which utilize multiplex beads coupled with single antigens. Hundreds of HLA antigens are identified to date, and the realization that antibody reactivity against the antigens is multispecific presented difficulties in accurately defining antibody specificity. Although Cross Reacting Groups (CREG), describing cross reactivity among HLA antigens, were helpful with determining specificity, they proved to be inadequate for the highly sensitized patients. Amino acid sequencing and three-dimensional modeling of the HLA molecules significantly advanced our understating of the HLA antigens and their epitopes. Although sensitive assays for antibody testing advanced analysis, they unmasked additional specificities undetectable by traditional methods, and the presence of naturally occurring anti-HLA antibodies in sera further complicated analysis and underscored the need to understand antibody reactivity and their epitopes. Hundreds of HLA class-I and class-II epitopes were defined by the Tekasaki and Duquesnoy groups and their usefulness in organ transplants were further advanced by a great number of transplant centers. Alloantibody specificities, CREGs, and nondonor specific antigens (NDSA) are now explained by public epitopes     

Acceptable HLA mismatches for highly immunized patients

Highly sensitized patients have developed antibodies against many different HLA antigens due to previous pregnancies, blood transfusions or failed transplants. These antibodies cause a positive crossmatch with almost all potential organ donors. As a positive crossmatch is a contra-indication for transplantation, highly sensitized patients have a low chance of transplantation unless special strategies are introduced. One such strategy is the acceptable mismatch program, which has led to transplantation of more than 300 of these highly sensitized patients within Eurotransplant. Centers are participating in the program on a voluntary basis. Before a patient can be included in this program, extensive antibody screening is necessary to define those HLA-A and -B antigens towards which the patient has never formed antibodies. Organ donor selection is based on complete compatibility with the patients own HLA antigens in combination with the acceptable mismatches. If such a combination is identified, mandatory exchange takes place. Despite the success of the acceptable mismatch program, about 25% of the patients will never receive a donor offer. These are patients with rare HLA antigens or rare combinations of HLA antigens. In the last few years, this group of patients has had the advantage of two additional programs running within Eurotransplant. In the HIT (highly immunized tray) program, sera of highly sensitized patients are sent to the different centers and crossmatched with all ABO compatible donors. In the case of a negative crossmatch, mandatory exchange takes place. Secondly, these patients can benefit from the extra points they receive for their waiting time, high antibody reactivity and rare HLA type in the standard Eurotransplant allocation system. We conclude that the application of these three strategies will lead to a significantly increased transplantation rate of highly sensitized patients.     

COVID-19 does not impact HLA antibody profile in a series of waitlisted renal transplant candidates

HLA antibodies are typically produced after exposure to transplanted tissue, pregnancy, and blood products. Sensitization delays access to transplantation and preclude utilization of donor organs. Infections and vaccinations have also been reported to result in HLA antibody formation. It is not known if patients develop HLA antibodies after infection with SARS-CoV-2. Here we analyzed a series of eighteen patients waiting for kidney transplantation who had symptomatic COVID-19 disease and recovered. None of the patients in this initial series developed de novo HLA antibodies. Notably, there was no increase in preexisting HLA antibodies in four highly sensitized patients with a CPRA > 80%. These preliminary data suggest that there may not be a need to repeat HLA antibody testing or perform a physical crossmatch on admission serum before kidney transplant for COVID-19 recovered patients. Data from a large number of patients with different demographics needed.     

HLA class I epitopes: recognition of binding sites by mAbs or eluted alloantibody confirmed with single recombinant antigens

Development of beads coated with single recombinant HLA antigens has permitted the confirmation and further definition of HLA class I epitopes. In this study, monoclonal antibodies (mAbs) or alloantibodies eluted from recombinant cell lines were tested for reactivity with Luminex beads individually coated with 79 recombinant HLA class I single antigen (rHLA SA). Published amino acid sequences were used to map epitopes common to sets of antigens reactive with each antibody. While several epitopes have already been demonstrated, this study confirmed them by adsorption of allosera with transfectants or SA beads having a single HLA antigen and specific binding of the eluted antibody on SA beads. The allosera and mAbs used in this study recognized a total of at least 58 HLA class I epitopes, as demonstrated by their different adsorption/reactivity patterns. Of these, 25 epitopes were characterized by a single unique common amino acid, 30 shared 2 signature amino acids in close proximity, and 3 epitopes involved 3 specific amino acids in a non-linear sequence. Since these epitopes may be targets for antibody-mediated allograft rejection, epitope analysis should complement HLA and CREG assignment for defining complex antibodies and identifying suitable donors for highly sensitized transplant patients.     

A broad and strong humoral immune response to donor HLA after implantation of cryopreserved human heart valve allografts

Cryopreserved human heart valves are used for valve replacement in patients with congenital or acquired heart disease. Although no blood group or human leukocyte antigens (HLA) matching is performed and no immunosuppression is administered, the clinical results are relatively good. After valve replacement, the majority of the patients develop HLA antibodies, whereas a smaller group of patients shows valve-related events at the long term after right ventricular outflow tract reconstruction. Therefore, we hypothesized that not the mere presence, but rather the titers of antidonor HLA antibodies may be related to valve allograft failure. The presence and specificity of HLA class I antibodies were determined by complement-dependent microlymphocytotoxicity (CDC) test in longitudinally taken peripheral blood samples of 35 valve allograft recipients. In eight patients with an antibody response specific against donor-HLA class I, the titers were measured by this CDC method after stepwise dilution of the plasma. Panel reactive antibodies of more than 10% were found in 31 of 35 (89%) valve allograft recipients. From these 31 patients, 24 (77%) developed donor-specific HLA class I antibodies. All eight selected patients had detectable donor-specific antibody titers, ranging from 1:2 to 1:8,000. Two donor valve recipients before retransplantation had (donor-specific) HLA antibodies and showed high titers of 1:256 and 1:8,000 shortly after the second allograft valve replacement, which was associated with an early graft failure in the latter patient. We conclude that transplantation of cryopreserved human heart valve allografts leads to a broad and strong humoral response, which is probably the result of a lack of immunosuppressive therapy after valve transplantation. Patients receiving a second or following valve allograft appeared to be sensitized and developed early and high allo-antibody titers after second valve allograft implantation. Valve failure was diagnosed in a patient with extremely high titers. These findings suggest that preoperative cross-matching may identify patients with high donor-specific HLA antibody titers and may reduce the risk for early recurrent graft failure.     

Pre-exposure to sub-saturating concentrations of HLA class I antibodies confers resistance to endothelial cells against antibody complement-mediated lysis by regulating Bad through the phosphatidylinositol 3-kinase/Akt pathway

Allografts transplanted across HLA-sensitization results in an antibody-mediated rejection known as hyperacute rejection. Depleting anti-graft antibodies from the recipient by plasmapheresis prior to transplantation can prevent this rejection. We developed an in vitro model using polyclonal HLA class I antibodies obtained from highly sensitized patients awaiting transplantation,and analyzed their ability to provide signals following binding to human aortic endothelial cells (EC). Using this model, we show that EC undergo caspase 3-dependent cell death by apoptosis upon exposure to saturating concentrations of HLA class I antibodies and complement accompanied by loss of Akt activation and phosphorylation of Bad. In contrast, exposure of EC to sub-saturating concentrations of HLA class I antibodies conferred resistance towards antibody/complement-mediated lysis termed accommodation. Accommodated EC exhibited reduction in the expression of the adhesion molecules ICAM-1 and VCAM-1 and a significant increase in the expression of anti-apoptotic genes Bcl-xL, Bcl-2 and heme oxygenase-1. Further, induction of phosphatidylinositol 3-kinase (PI3K) and Akt activities that facilitate the phosphorylation of Bad were also noted. In conclusion, exposure of sub-saturating concentrations of HLA class I antibodies results in the induction of PI3K/Akt pathway that confers resistance to endothelial cells against antibody/complement-mediated cell death.     

The ABCs (DRDQDPs) of the prozone effect in single antigen bead HLA antibody testing: Lessons from our highly sensitized patients

Accurate identification of HLA antibodies using the single antigen bead (SAB) assay is critical for assessment of pre/post-transplant immunological risk and successful virtual crossmatching. Unfortunately, high titer HLA antibodies can be missed or underestimated in the SAB assay as a result of interference with the detection of IgG. This so called prozone effect has been attributed to both complement- and IgM-dependent mechanisms and can be minimized with serum dilution or treatment with heat, EDTA, or DTT. In this study we describe the frequency, nature, and degree of prozone in a cohort of highly sensitized patients (cPRA ≥ 95%), in whom accurate detection of HLA antibodies and virtual crossmatching is of paramount importance. Sera were tested by the SAB assay ± EDTA treatment, ±1:10 dilution to identify the prozone effect. The relative contribution of complement vs IgM to prozone was assessed using anti-C3d and anti-IgM reporter antibodies, respectively. We found that prozone was very frequent in highly sensitized patients (80%), especially those with a history of previous transplantation (87%). Class I HLA specificities were more commonly affected than class II and the susceptibility to prozone was locus dependent with HLA-A(31%), -B(29%) and -DQ(26%) being affected more frequently than HLA-DP(17%), -C(16%) and -DR(5%) antigens. Interestingly, the presence of prozone could be predicted by C3d positivity (MFI ≥ 4000; sensitivity = 95.2%, specificity = 97.2%) and the degree of prozone correlated directly with the extent of C3d deposition. The role of IgM was less clear. However, serum dilution studies suggested that IgM may contribute to interference in a small subset of prozone positive specificities. Our study underscores the importance of serum treatment to inhibit complement activation and minimize prozone in the SAB assay, especially in highly sensitized patients.     

Kidney transplantation of highly sensitized recipients under the new kidney allocation system: A reflection from five different transplant centers across the United States

Deceased donor kidney allocation was reorganized in the United States to address several problems, including the highly sensitized patients disadvantaged with large, diverse repertoires of antibodies. Here, five transplant surgeons review their center’s experience with the new allocation changes: highlighting areas of accomplishment, opportunities for improvement and, in some cases, stark differences in practice. Across these five centers the highly sensitized patients (CPRA ?98%) range from 5.5 to 9.2% of the 12,364 candidates on their collective waitlist. All centers reported greater rates of kidney transplantations in highly sensitized patients (12.4–27%). Three of the programs (Emory, UCSF, UW) relied upon the virtual crossmatch prior to organ acceptance in a majority of cases (70–86%)—the mere presence of antibody on HLA antibody screen was sufficient to exclude the donor in most cases at Emory and UCSF. Penn and UAB relied upon the physical flow crossmatch in almost all cases prior to proceeding with transplantation. Current or historical donor-specific antibody was occasionally crossed in certain cases at UW and UAB necessitating IVIG/plasmapheresis and/or B cell depletion perioperatively. Some authors raised concerns for cost efficiency given the increased need for organ/specimen transportation, and extensive use of hospital resources and ancillary services. In general, we found that the new allocation system has successfully achieved one of its primary goals—increased kidney transplantation in the disadvantaged, highly sensitized patients; the long-term outcomes in all patients and the cost ramifications of these changes will require continued reassessment and clarification.     

复发性流产患者HLA及血小板特异性糖蛋白抗体GPⅡb/Ⅲa表达的初步研究

目的:探讨原因不明复发性流产(RSA)患者HLA抗体和血小板特异性糖蛋白(GPⅡb/Ⅲa)抗体的表达情况。方法:对299名复发性流产的患者血浆中HLA抗体和GPⅡb/Ⅲa抗体进行检测,血小板抗体检测试剂盒(固相凝集法)筛查IgG抗体,Antigen Tray-LATM板检测HLA抗体,GTI公司PAK-PLUS检测试剂盒检测血小板特异性抗体,利用流式细胞仪技术进行GPⅡb/Ⅲa抗体的确证。结果:检测的299例RSA患者中,IgG抗体阳性26例,阳性率8.69%。26例IgG抗体阳性患者中,25例HLA抗体呈阳性。HLA-Ⅰ类抗体阳性占5.02%,HLA-Ⅱ类抗体阳性占1.33%,HLA-Ⅰ+Ⅱ类抗体阳性占2.01%;在26例RSA患者中,GPⅡb/Ⅲa抗体阳性4例(占15.38%),其中GPⅡb/Ⅲa阳性和HLA抗体均阳性3例(占11.54%),GPⅡb/Ⅲa抗体单独阳性1例(占3.85%)。结论:初步探讨了RSA患者HLA抗体和GPⅡb/Ⅲa抗体的分布情况,本研究为复发性流产患者血小板抗体的检测提供了初步的实验基础。