HLA class II DQA and DQB epitopes: Recognition of the likely binding sites of HLA-DQ alloantibodies eluted from recombinant HLA-DQ single antigen cell lines

Donor-specific antibodies (DSA) in sera of sensitized transplant patients are often produced against the specific epitopes on mismatched HLA antigens. In this study, we selected sera from 30 kidney transplant patients with DSA and AMR to define DQ epitopes. Using adsorption and elution assays, we identified 18 antibody reaction patterns to define 6 new epitopes and to confirm 12 previously defined epitopes. In one patient case, one mismatched antigen produced 3 different antibodies and, in another, antibodies were produced against the alpha and beta chains of the same antigen. For some sera, a single epitope can explain reactions for 27 of the 29 DQ beads in the single antigen panel.     

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.     

HLA antibody identification with single antigen beads compared to conventional methods

Single antigen (SA) beads coated with Class I HLA antigens from recombinant cells lines were tested with 170 mouse monoclonal antibodies (mAbs). The HLA specificities of all mAbs were previously determined by the cytotoxicity assay (CDC). There were 100 mAbs which produced the expected reactions with the SA beads, indicating that the SA beads coated with the antigens had reacted properly. Sixty one mAbs were positive with one or more antigen(s) that shared unique amino acids (aa) possibly constituting a common epitope. Single antigen beads were then tested on 58 alloantisera analyzed by 63 laboratories of the UCLA serum exchange (UCLA-SE). Many specificities detected by the single antigen beads were missed by the laboratories employing conventional methods. Most of the missed specificities were of lower frequency, although in some instances, even common specificities were missed. These findings have important implications regarding the use of specificities to predict positive crossmatch, to selecting platelet donors for highly sensitized recipients, and analysis of sera for donor specific antibodies.     

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.     

HLA Histocompatibility Antigens in a Polynesian Population — Cook Islanders of Mauke

Polynesians living on the island of Mauke in the Cook Island group were typed for HLA-A and -B locus antigens. The Mauke population has restricted HLA polymorphism, with five A-locus antigens and four B-locus antigens accounting for a majority of the HLA phenotypes. Although some differences in antigen frequency were found when Mauke Islanders were compared with Polynesians from Easter Island and Samoa, the Mauke Islanders were closer in their HLA antigenic profile to polynesians than to Melanesians.     

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.     

Epitope-specificities of HLA antibodies: The effect of epitope structure on Luminex technique-dependent antibody reactivity

The search of HLA antibodies is currently more accessible by solid-phase techniques (Luminex) in the immunized patients leading to an expansion of the antibody patterns. The aim of this study was to investigate low median fluorescence intensity value in unexpected reactivity patterns. Here, we performed HLAMatchmaker analyses to evaluate the potential functional epitopes that can elicit HLA-specific alloantibody responses in a pregnancy-sensitized woman with an epitope defined by the 82LR. Surprisingly, in according to the registry of HLA epitopes, we found that 82LR epitope covered all allelic specificities of our unexpected antibody patterns, shared between Bw4-positive HLA-B antigen and HLA-A23, -A24, -A25 and -A32. This finding is consistent with the verification of HLA ABC epitope recorded in the website-based HLA Epitope Registry and addresses the importance of determining HLA antibody epitope-specificities on Luminex technique-dependent antibody reactivity.     

Correlations between Terasaki's HLA class II epitopes and HLAMatchmaker-defined eplets on HLA-DR and -DQ antigens

Human leukocyte antigen (HLA) class II-specific antibodies increase the risk of transplant failure, and their characterization must consider epitopes rather than antigens. There are two strategies to determine HLA epitope structure. Terasaki's group has analyzed antibody reactivity patterns with single antigen panels with a computer program based on shared amino acid residues of reactive alleles. HLAMatchmaker is a theoretical algorithm that predicts HLA epitopes on the HLA molecular surface from stereochemical modeling of epitope–paratope interfaces of antigen–antibody complexes. Our epitope repertoire is based on so-called 'eplets' representing 3-Å patches of at least one polymorphic residue on the molecular surface. This report describes how 49 of 53 Terasaki's HLA-DR epitopes correspond to HLAMatchmaker-defined eplets. Most of them are equivalent to single eplets ( n  = 33) or two or more possible eplets ( n  = 10), but six had corresponding eplet pairs. There were 10 cases whereby eplets have permissible residue combinations, and in 5 cases, we found that eplet specificity might be influenced by nearby hidden residues. We could assign corresponding eplets to 17 of 18 Terasaki's HLA-DQ epitopes. This study demonstrates how the HLAMatchmaker interpretation of amino acid residues shared between antibody-reactive antigens can increase our understanding of the structural basis of HLA epitopes.     

Shared cadaver donor-husband HLA class I mismatches as a risk factor for renal graft rejection in previously pregnant women

During the last few years, we have observed four cases in which accelerated rejection of a cadaver donor kidney in a previously pregnant woman could be clearly attributed to the rapid emergence of anti-human leukocyte antigen (HLA) antibodies that had been stimulated by mismatched paternal antigens but were completely undetectable at the time of transplantation. In addition to reviewing those cases, we also reviewed data on 19 other women with a history of at least one pregnancy who underwent transplantation with a first cadaveric kidney since 1991 and were followed for at least six months. The HLA antigens of the husbands had to have been determined and all accelerated rejection or early graft losses due to confirmed or presumed immunological causes were considered. Of the 19 additional women meeting these inclusion criteria, three suffered early immunological graft loss. As in our index cases, two of these women had also received kidneys from donors who shared at least one major immunogenic mismatched antigen with the respective husband for a total of six of seven women with early immunological graft loss. Only one of the 16 women without accelerated rejection or early immunological graft loss had a donor who shared a mismatched antigen with her husband. The difference between the two groups is statistically significant (p = 0.0005). These findings, considered with individual cases reported by other groups, indicate that transplantation from a cadaver donor with immunogenic mismatched class I HLA antigen(s) shared with the husband should be avoided in women with a previous history of pregnancy even when anti-HLA antibodies are not currently detected.     

The HLA-DR phenotype of the responder is predictive of humoral response against HLA class I antigens

Recent studies suggest that the immunogenicity of an human leukocyte antigen (HLA) incompatibility should be considered in the context of the HLA phenotype of the recipient. The HLA-DR phenotype of the responder is thought to be predictive for the strength of the alloimmune response. In order to analyze the humoral response against HLA class I antigens in the context of the HLA-DR phenotype of the responder, we selected all HLA-DR homozygous Dutch patients that were present on the Eurotransplant waiting list between 1967 and 2000 (n=1,317 patients). By logistic regression it was determined whether antibody production against a specific HLA class I antigen is associated with a particular HLA-DR antigen in the patient. Furthermore, it was analyzed whether a patient, expressing a particular HLA-DR antigen, preferentially produces antibodies against particular HLA class I antigens. The results demonstrate that patients, homozygous for a certain HLA-DR antigen, cannot be considered high or low responders when analyzing the antibody response in terms of panel reactive antibody (PRA) value. However, a correlation can be found between the HLA-DR phenotype of the patient and the specific antibody response against HLA class I antigens. For example, antibodies against HLA-A10, -A11, -A19, and -B35 are produced more frequently by HLA-DR6 positive individuals, whereas antibodies against HLA-A3, -B5, -B7, -B8, and -B12 are produced more frequently by HLA-DR4 positive individuals. These data confirm that the HLA-DR phenotype of the responder plays a determinative role in the immunogenicity of mismatched HLA antigens. The results indicate that selection of HLA class I mismatches of the donor in the context of the HLA-DR phenotype of the responder might reduce the incidence of humoral graft rejection and minimize the sensitization grade of retransplant candidates.     

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.     

Human monoclonal HLA antibodies reveal interspecies crossreactive swine MHC class I epitopes relevant for xenotransplantation

Crossreactivity of anti-HLA antibodies with SLA alleles may limit the use of pig xenografts in some highly sensitized patients. An understanding of the molecular basis for this crossreactivity may allow better selection of xenograft donors. We have tested 68 human monoclonal HLA class I antibodies (mAbs) for reactivity with pig lymphocytes from SLA defined pigs and found nine to be crossreactive. Eight of nine were broadly HLA reactive IgM-mAbs. The putative HLA epitopes for seven mAbs. were conserved in the aminoacid sequence of the SLA alleles studied. The lack of reactivity of a large number of mAbs largely correlated with the absence of the putative epitopes in the SLA alleles studied. We conclude that most patients with anti-HLA class I antibodies should be able to find pig donors lacking SLA antigens that cross react with their antibodies and that many of the crossreacting epitopes can be defined by analysis of shared epitopes in the aminoacid sequence of human and pig MHC antigens.     

Alloimmunization to public HLA antigens in multi-transfused platelet recipients

Multitransfused recipients of random donor platelet concentrates frequently form broadly-reactive human leukocyte antigen (HLA) antibodies which restrict the pool of suitable donors to those who are HLA matched with the recipient. In an effort to expand the donor pool for such patients, a retrospective analysis of the antibodies formed by alloimmunized recipients was performed taking sensitization to public HLA antigens into account. A total of 144 sera from 18 subjects was analyzed. Peak reactive sera from these patients ranged from 53 to 100 percent when tested against lymphocyte panels. When the reaction patterns of these sera were evaluated, 11 patients formed antibodies against public antigens only, three patients formed antibodies reacting exclusively with private determinants, and two patients formed antibodies reacting with both public and private antigens. The specificities in highly reactive sera from two patients could not be identified. A total of 153 lymphocyte crossmatches was performed while these patients were receiving single donor HLA-matched platelets. Forty-six positive crossmatches were obtained of which 33 occurred with donors mismatched for at least one non-crossreactive HLA antigen with the recipient. Of these, 26/33 (79 percent) positive crossmatches were predicted by the results of the serum analysis for antibodies directed against public HLA determinants. Similarly, 31/40 (78 percent) negative lymphocyte crossmatches were predictable on the basis of the serum analysis. It is concluded that "multispecific" lymphocytotoxic antibodies formed by multitransfused recipients of platelet concentrates, are often directed against one or a few public HLA determinants.(ABSTRACT TRUNCATED AT 250 WORDS)     

The isolation and characterization of murine monoclonal antibodies directed to polymorphic epitopes on HLA antigens

Twenty-two murine monoclonal antibodies directed to distinct polymorphic epitopes on HLA class I or class II antigens have been isolated and characterized using a simple protocol for fusion and hybridoma selection. Thirteen MAbs directed to class I antigens are reported here for the first time. The majority of these MAbs reacted with multiple specificities, often revealing a surprising sharing of epitopes. MAbs directed against single classically defined alloantigenic specificities were also obtained.     

Inhibition of HLA B8-restricted recognition by unrelated peptides: evidence for allosteric inhibition

A panel of synthetic peptides representing human lymphocyte antigen (HLA) B8, other class I and class II restricted T cell epitopes and two B cell epitopes, were all able to compete with recognition of a HLA B8 restricted epitope by a cytotoxic T cell clone. Competition was obtained when the competitor peptides were added either before or after the target epitope. The target epitope also had a slow off rate, implicating allosteric inhibition. The presence of non-specific, allosteric binding sites may interfere with experiments attempting to define immunologically relevant MHC binding specificities.     

A reverse-engineering strategy utilizing the integration of single antigen beads and NGS HLA genotypes to detect potential antibody inducing epitopes

Central to the idea of antibody recognition is some degree of foreignness of the target antigen compared to the antibody producer. Epitopes are distinct regions on an antigen to which antibody can be elicited and bound. However, for HLA antigens, there is no consensus definition of what represents the minimal functional immunogenic unit of dissimilarity. To assess this in an unbiased way, we developed a reverse engineering software strategy based on donor specific antibodies defined by single antigen beads and full length genomic high resolution HLA typing by NGS of recipients and donors (332 transplant pairs). Starting with the ATG of Exon 1 and moving stepwise one amino acid at a time for each of the following triplets, the algorithm compared every possible amino acid triplet of the recipient and donor for 11 loci (A, B, C, DRB1, DRB3, DRB4, DRB5, DQA1, DQB1, DPA1, DPB1). Results were agnostic with respect to HLA class, not restricted to just the mature protein, and not influenced by existing maps (e.g., IMGT, or epitope models). We also developed web-based functions in the 17th IHIWS database to collect the unbiased triplets so that we could group the transplant pairs with the same donor specific antibodies and find shared triplets within the groups as potential core or essential epitopes that trigger the antibody formation. Profiling the pairs where the same DSA was identified led to identification of discrete amino acid triplets shared among the pairs irrespective of HLA match. The potential epitopes were mapped onto the 3D protein structure for reference.     

Epitope specificity of HLA class I alloantibodies I. Frequency analysis of antibodies to private versus public specificities in potential transplant recipients

Sera obtained sequentially from 419 patients awaiting solid organ transplantation were screened and analyzed for HLA class I epitope specificity. Antibodies detected in each serum were defined as “private” if reactivity could only be demonstrated against a single specificity within one of the eight major CREGs, or as “public” if reactivity in a serum could be demonstrated against two or more specificities within a single CREG. A total of 139 sera contained % PRA > 0, in which 147 specific antibodies were identified. Of the 103 positive sera, 93 (90%) contained antipublic antibodies, with or without additional antiprivate antibodies, whereas just 10 (10%) sera contained only apparent antiprivate antibodies. The success rate in defining antibody specificities was low at PRA values of 1%–20% due to weak reactivity and high false-positive rates. Specificity analysis with high test sensitivity and specificity was achieved with PRA values between 40% and 80%. At PRA values > 80%, test sensitivity remained high but specificity declined. We conclude that most anti-HLA antibodies are directed against high frequency public epitope clusters (CREGs), and highly sensitized patients develop antibodies in a fairly predictable fashion, a feature that significantly improved the success rate of specificity analysis. Since high frequency antipublic antibodies are common sequelae of CREG mismatches, further definition of HLA class I public epitopes eventually may be important in donor-recipient matching.     

Predicted indirectly recognizable HLA epitopes presented by HLA-DR correlate with the de novo development of donor-specific HLA IgG antibodies after kidney transplantation

Background

HLA class-I mismatches selectively induce antibody formation after kidney transplantation. The de novo development of donor-specific IgG HLA class-I antibodies may be dependent on the HLA class-II background of the patient by presenting T-helper epitopes within the recognized HLA class-I antigens.

Methods

The correlation between antibody production against mismatched donor human leukocyte antigens (HLA) class I and the number of HLA class II-restricted predicted indirectly recognizable HLA epitopes (PIRCHE-II) in the respective HLA class-I mismatches was investigated. To this end, we analyzed sera taken after nephrectomy from a cohort of 21 non-immunized individuals that received a renal transplant.

Results

Fourty-nine HLA class-I mismatches were found which all contained immunogenic eplets according to HLAMatchmaker. Donor specific HLA antibody responses were detected against 38 HLA class-I mismatches after nephrectomy. These mismatches were found to contain a larger number of PIRCHE-II when compared to mismatches which did not induce donor specific HLA antibodies. Most PIRCHE-II (68%) were not part of an eplet as defined by HLAMatchmaker.

Conclusions

Our data suggest that presentation of donor-derived HLA class-I peptides by recipient HLA class-II molecules plays a significant role in de novo development of donor-specific IgG HLA antibodies.