HLA Epitope Registry Research Articles

An Approach to Identify HLA Class II Immunogenic Epitopes in the Greek Population through Machine Learning Algorithms

Current pre-transplantation routine matching involves serum anti-HLA antibodies quantification but cannot always preclude unfavorable graft outcomes. Epitope-based matching is proposed as a more precise approach, but to date no epitope-matching algorithm provides a satisfactory predictive tool for transplantation outcomes. In this study, anti-HLA-II loci responses from 1748 patients were analyzed with unsupervised machine learning algorithms, namely principal component analysis (PCA) and antigenic distances, projected as dendrograms. PCA for anti-HLA-DR anti-bodies revealed three main clusters of responses: anti-HLA-DR51 combined with anti-HLA-DRB1*01, anti-HLA-DR52 combined with anti-HLA-DRB1*08 and anti-HLA-DR53 combined with anti-HLA-DRB1*10. The dendrogram for anti-HLA-DR confirmed the pattern and showed further bisection of each cluster. Common epitopes present exclusively in all HLA molecules of each cluster were determined following the HLA epitope registry. Thus, we propose that 19 out of 123 HLA-DR epitopes are those that mainly lead anti-HLA-DR responses in the studied population. Likewise, we identified 22 out of 83 epitopes responsible for anti-HLA-DQ and 13 out of 62 responsible for anti-HLA-DP responses. Interpretation of these results may elucidate mechanisms of interlocus cross-reactivity, providing an alternative way of estimating the significance of each epitope in a population and thus suggesting a novel strategy towards optimal donor selection.

Eplet-Predicted Antigens: An Attempt to Introduce Eplets into Unacceptable Antigen Determination and Calculated Panel-Reactive Antibody Calculation Facilitating Kidney Allocation.

(1) Calculated panel-reactive antibody (CPRA) is a measure of sensitization based on unacceptable antigens (UAs). Determination of UAs based on single-antigen bead assays at allele or antigen levels may be inappropriate. We aimed to introduce eplets for better assessment of sensitization; (2) 900 recipients and 1427 donors were enrolled for candidate or donor pools, respectively. Eplets were from the HLA Epitope Registry. UAs were determined by anti-HLA antibodies identified using LIFECODES Single Antigen (LSA) kits. CPRA values were calculated using a simplified method of donor filtering; (3) HLA antigens containing all eplets of an HLA antigen in LSA kits (LSA antigen) were defined as eplet-predicted (EP) antigens, the reactivity of which could be predicted by that LSA antigen. High reactivity concordance was found between LSA and EP antigens. More HLA antigens were covered by EP antigens in the population than LSA antigens. CPRA values at the EP level were higher than at the allele level and lower than at the antigen level. The EP antigens facilitated UA determination for non-LSA antigens and avoided acute rejection; (4) UA determination using EP antigens can lead to more accurate assessment of sensitization, enabling a high probability of compatible organs and a low risk of adverse outcomes.

Site-directed mutagenesis of HLA molecules reveals the functional epitope of a human HLA-A1/A36-specific monoclonal antibody.

Eplet 44KM is currently listed in the HLA Epitope Registry but does not adhere to the eplet definition of an amino acid configuration within a 3.5Å radius. Eplet 44KM has been previously redefined to the antibody-verified reactivity pattern 44K/150V/158V, based on reactivity analysis of monoclonal antibody VDK1D12. Since the three residues are always simultaneously present on common HLA alleles, methods to define which residue is crucial for antibody-induction and binding are limited. In this proof-of-concept study, we performed site-directed mutagenesis to narrow down the antibody-verified reactivity pattern 44K/150V/158V to a single amino acid and defined 44K as the eplet or functional epitope of mAb VDK1D12.

Snowflake: A deep learning-based human leukocyte antigen matching algorithm considering allele-specific surface accessibility.

Histocompatibility in solid-organ transplantation has a strong impact on long-term graft survival. Although recent advances in matching of both B-cell epitopes and T-cell epitopes have improved understanding of allorecognition, the immunogenic determinants are still not fully understood. We hypothesized that HLA solvent accessibility is allele-specific, thus supporting refinement of HLA B-cell epitope prediction. We developed a computational pipeline named Snowflake to calculate solvent accessibility of HLA Class I proteins for deposited HLA crystal structures, supplemented by constructed HLA structures through the AlphaFold protein folding predictor and peptide binding predictions of the APE-Gen docking framework. This dataset trained a four-layer long short-term memory bidirectional recurrent neural network, which in turn inferred solvent accessibility of all known HLA Class I proteins. We extracted 676 HLA Class-I experimental structures from the Protein Data Bank and supplemented it by 37 Class-I alleles for which structures were predicted. For each of the predicted structures, 10 known binding peptides as reported by the Immune Epitope DataBase were rendered into the binding groove. Although HLA Class I proteins predominantly are folded similarly, we found higher variation in root mean square difference of solvent accessibility between experimental structures of different HLAs compared to structures with identical amino acid sequence, suggesting HLA’s solvent accessible surface is protein specific. Hence, residues may be surface-accessible on e.g. HLA-A*02:01, but not on HLA-A*01:01. Mapping these data to antibody-verified epitopes as defined by the HLA Epitope Registry reveals patterns of (1) consistently accessible residues, (2) only subsets of an epitope’s residues being consistently accessible and (3) varying surface accessibility of residues of epitopes. Our data suggest B-cell epitope definitions can be refined by considering allele-specific solvent-accessibility, rather than aggregating HLA protein surface maps by HLA class or locus. To support studies on epitope analyses in organ transplantation, the calculation of donor-allele-specific solvent-accessible amino acid mismatches was implemented as a cloud-based web service.

A Comprehensive Evaluation of the Antibody-Verified Status of Eplets Listed in the HLA Epitope Registry.

Matching strategies based on HLA eplets instead of HLA antigens in solid organ transplantation may not only increase the donor pool for highly sensitized patients, but also decrease the incidence of de novo donor-specific antibody formation. However, since not all eplets are equally capable of inducing an immune response, antibody verification is needed to confirm their ability to be bound by antibodies, such that only clinically relevant eplets are considered. The HLA Epitope Registry has documented all theoretically defined HLA eplets along with their antibody verification status and has been the foundation for many clinical studies investigating eplet mismatch in transplantation. The verification methods for eplets in the Registry range from polyclonal sera from multi- and uni-parous women to murine and human monoclonal antibodies (mAbs), and antibodies purified by adsorption and elution from sera of HLA immunized individuals. The classification of antibody verification based on different methods for validation is problematic, since not all approaches represent the same level of evidence. In this study, we introduce a classification system to evaluate the level of evidence for the antibody-verified status of all eplets in the HLA Epitope Registry. We demonstrate that for a considerable number of eplets, the antibody-verified status is solely based on polyclonal serum reactivity of multiparous women or on reactivity of murine mAbs. Furthermore, we noted that a substantial proportion of patient sera analyses and human mAb data presented in the HLA Epitope Registry Database has never been published in a peer-reviewed journal. Therefore, we tested several unpublished human HLA-specific mAbs by luminex single antigen beads assay to analyze their HLA reactivity for eplet antibody verification. Although the majority of analyzed mAbs indeed verified their assigned eplets, this was not the case for a number of eplets. This comprehensive overview of evidence for antibody verification of eplets in the HLA Epitope Registry is instrumental for future investigations towards eplet immunogenicity and clinical studies considering antibody-verified eplet mismatch in transplantation and warrants further standardization of antibody verification using high quality data.

HLA-EPI: A new EPIsode in exploring donor/recipient epitopic compatibilities.

The HLA system plays a pivotal role both in transplantation and immunology. While classical HLA genotypes matching is made at the allelic level, recent progresses were developed to explore antibody–antigen recognition by studying epitopes. Donor to recipient matching at the epitopic level is becoming a trending topic in the transplantation research field because anti‐HLA antibodies are epitope‐specific rather than allele‐specific. Indeed, different HLA alleles often share common epitopes. We present the HLA‐Epi tool (hla.univ-nantes.fr) to study an HLA genotype at the epitope level. Using the international HLA epitope registry (Epregistry.com.br) as a reference, we developed HLA‐Epi to easily determine epitopic and allelic compatibility levels between several HLA genotypes. The epitope database covers the most common HLA alleles (N = 2976 HLA alleles), representing more than 99% of the total observed frequency of HLA alleles. The freely accessible web tool HLA‐Epi calculates an epitopic mismatch load between different sets of potential recipient‐donor pairs at different resolution levels. We have characterized the epitopic mismatches distribution in a cohort of more than 10,000 kidney transplanted pairs from European ancestry, which showed low number of epitopic mismatches: 56.9 incompatibilities on average. HLA‐Epi allows the exploration of epitope pairing matching to better understand epitopes contribution to immune responses regulation, particularly during transplantation. This free and ready‐to‐use bioinformatics tool not only addresses limitations of other related tools, but also offers a cost‐efficient and reproducible strategy to analyze HLA epitopes as an alternative to HLA allele compatibility. In the future, this could improve sensitization prevention for allograft allocation decisions and reduce the risk of alloreactivity.

Hidden Patterns of Anti-HLA Class I Alloreactivity Revealed Through Machine Learning.

Detection of alloreactive anti-HLA antibodies is a frequent and mandatory test before and after organ transplantation to determine the antigenic targets of the antibodies. Nowadays, this test involves the measurement of fluorescent signals generated through antibody–antigen reactions on multi-beads flow cytometers. In this study, in a cohort of 1,066 patients from one country, anti-HLA class I responses were analyzed on a panel of 98 different antigens. Knowing that the immune system responds typically to “shared” antigenic targets, we studied the clustering patterns of antibody responses against HLA class I antigens without any a priori hypothesis, applying two unsupervised machine learning approaches. At first, the principal component analysis (PCA) projections of intra-locus specific responses showed that anti-HLA-A and anti-HLA-C were the most distantly projected responses in the population with the anti-HLA-B responses to be projected between them. When PCA was applied on the responses against antigens belonging to a single locus, some already known groupings were confirmed while several new cross-reactive patterns of alloreactivity were detected. Anti-HLA-A responses projected through PCA suggested that three cross-reactive groups accounted for about 70% of the variance observed in the population, while anti-HLA-B responses were mainly characterized by a distinction between previously described Bw4 and Bw6 cross-reactive groups followed by several yet undocumented or poorly described ones. Furthermore, anti-HLA-C responses could be explained by two major cross-reactive groups completely overlapping with previously described C1 and C2 allelic groups. A second feature-based analysis of all antigenic specificities, projected as a dendrogram, generated a robust measure of allelic antigenic distances depicting bead-array defined cross reactive groups. Finally, amino acid combinations explaining major population specific cross-reactive groups were described. The interpretation of the results was based on the current knowledge of the antigenic targets of the antibodies as they have been characterized either experimentally or computationally and appear at the HLA epitope registry.

A Novel Tool to Define the Immunogenicity of HLA Mismatches

In kidney transplantation, it is pivotal to prevent HLA donor specific antibody (DSA) formation, which is triggered by immunogenic epitopes present on mismatched HLA. The optimal donor can theoretically be selected by avoiding these epitope mismatches. To implement epitope matching, it is essential to define the relevant epitope mismatches between recipient and donor. Therefore, we aimed at developing and validating a tool to define amino acid mismatches on donor HLA. This tool can be used to define the immunogenicity of HLA mismatches for a large number of recipient-donor pairs. We developed a computer algorithm containing all HLA amino acid (AA) sequences from the IPD-IMGT/HLA database and all theoretical and antibody-verified eplet AA (position and type) from the HLA Epitope Registry. The HLA input is preferably second field typing, as the HLA sequences of the recipient are compared to each mismatched HLA allele of the donor to define the number of AA mismatches. In addition, the physical properties of the AA mismatches are defined, which can be correlated to Luminex single antigen DSA formation after transplantation to define the immunogenic epitopes. This tool and other approaches (EpVix, Cambridge AA mm score) were used to analyse 233 first kidney transplant recipient-donor pairs as part of the 17th IHIWS epitope component. Comparing the results of all approaches similar number of mismatches were observed. For both HLA class I and class II, we found that increasing numbers of AA mismatches resulted in a higher probability of DSA formation (as indicated in figure 1). This newly developed computer algorithm can be used to define HLA AA mismatches for a large number of recipient-donor pairs from all kind of populations and to select optimal donors in kidney transplantation. An advantage of this tool is that all HLA alleles, including rare alleles, can be analysed.

Usefulness of the ElliPro epitope predictor program in defining the repertoire of HLA-ABC eplets

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.

P151 Acute antibody-mediated kidney rejection associated with anti-Bw6 originated from allosensitization to HLA-C14 during pregnancy: A case report

We report a case of a 36 year old Caucasian G4P4 female with chronic kidney disease stage 4 and no prior transfusions who received a living-related kidney transplant from her brother, which was a 0A-1B-1DR mismatch. She was discharged post-operative day 4 with good allograft function and a serum creatinine (SCr) of 0.76 mg/dL. Seven days later she presented with pain at the allograft site and a SCr of 3.6 mg/dL. Allograft biopsy showed evidence of thrombotic microangiopathy, hemorrhagic cortical necrosis, and peritubular capillary positivity for C4d. This strongly suggested an acute antibody mediated rejection (AMR), which was later corroborated by histology of the post-nephrectomy allograft specimen. HLA antibody screen at the time of AMR by single-antigen beads demonstrated a broad pattern of reactivity against all beads expressing Bw6-associated antigens and multiple HLA-C antigens. The MFI values of all Bw6-associated beads were above 15000 when tested at 1:25 dilution; MFI of the donor specific antibody against B62 (Bw6) was 19366. Only low-level reactivity against a small number of HLA-B antigens was detected prior to transplantation either with neat serum or at 1:25 dilution. These findings are consistent with an anamnestic alloantibody response against a common epitope present in HLA-B and HLA-C antigens, initially primed by exposure to her husband’s antigens during pregnancy and rechallenged by renal transplantation. HLA typing of the patient’s husband demonstrated no antigen mismatch at the A or B loci and one antigen mismatch at the C locus (patient is C16 and her husband is C14). Notably, C14 and all Bw6-associated antigens share a confirmed epitope, 80N (HLA epitope registry). This case demonstrates an unusual incidence of sensitization to Bw6-associated antigens via exposure to a Bw6-crossreactive C antigen. It also highlights the risk of occult alloimmunization that is undetectable in pre-transplant HLA antibody testing but may cause rapidly progressive allograft rejection.

Antibody-defined epitopes on HLA-DQ alleles reacting with antibodies induced during pregnancy and the design of a DQ eplet map

The concept that HLA antibodies recognize epitopes is leading to new approaches of HLA matching at the epitope level. HLA-DQ plays an important role and many studies have identified structurally defined DQ epitopes specifically recognized by antibodies; they have been recorded in the International HLA Epitope Registry http://www.epregistry.com.br but the list is still incomplete.Pregnancy offers an attractive model to study antibody responses to HLA epitopes. The current analysis was done on 42 DQ-reactive post-pregnancy sera tested in binding assays with a panel of DQ heterodimers. The reactivity of 29 sera corresponded fully to the presence of antibody-verified DQA and DQB epitopes recorded in the Registry. Analysis of the remaining 13 sera led to the identification of additional antibody-defined DQB and DQA epitopes.We have designed the first version of an eplet map for DQ alleles which includes antibody-defined DQA and DQB epitopes and shows sequence positions with polymorphic residues which can be used in HLA epitology studies to identify new antibody-defined DQ epitopes.

Detection of newly antibody-defined epitopes on HLA class I alleles reacting with antibodies induced during pregnancy.

The determination of HLA mismatch acceptability at the epitope level can be best performed with epitopes that have been verified experimentally with informative antibodies. The website-based International Registry of HLA Epitopes (http://www.epregistry.com.br) has a list of 81 antibody-verified HLA-ABC epitopes but more epitopes need to be added. Pregnancy offers an attractive model to study antibody responses to mismatched HLA epitopes which can be readily determined from the HLA types of child and mother. This report describes a HLAMatchmaker-based analysis of 16 postpregnancy sera tested in single HLA-ABC allele binding assays. Most sera reacted with alleles carrying epitopes that have been antibody-verified, and this study focused on the reactivity of additional alleles that share other epitopes corresponding to eplets and other amino acid residue configurations. This analysis led in the identification of 16 newly antibody-defined epitopes, seven are equivalent to eplets and nine correspond to combinations of eplets in combination with other nearby residue configurations. These epitopes will be added to the repertoire of antibody-verified epitopes in the HLA Epitope Registry.

Identification of epitopes on HLA-DRB alleles reacting with antibodies in sera from women sensitized during pregnancy

This report describes a HLAMatchmaker-based antibody analysis of post-pregnancy sera with antibodies against child-specific HLA-DR epitopes. These sera were reactive in IgG-binding assays with single allele bead (SAB) panels on a Luminex platform. The antibody specificity analysis focused on DRB epitopes that have been recorded in the International HLA Epitope Registry (http://www.epregistry.com.br) as experimentally verified with informative antibodies but we also considered other eplets that predict potential epitopes.The SAB panel has in several instances two or more alleles corresponding to the same serologically defined DR antigen and we selected six sera were with different reactivity patterns with DR1, DR4, DR13 and/or DR52 alleles. We demonstrate here how amino acid differences between these alleles can provide useful information in the determination of new epitope specificities of antibodies in these sera. Eight newly antibody-verified epitopes were identified including three that correspond to eplets paired with self-residue configurations. Epitope specificity information appears to be useful in the prediction of mismatch acceptability of non-SAB alleles within serological DR antigen groups.

EpViX: A cloud-based tool for epitope reactivity analysis and epitope virtual crossmatching to identify low immunologic risk donors for sensitized recipients

One of the challenges facing solid organ transplantation programs globally is the identification of low immunological risk donors for sensitized recipients by HLA allele genotype. Because recognition of donor HLA alleles by host antibodies is at the core of organ rejection, the objective of this work was to develop a new version of the EpHLA software, named EpViX, which uses an HLAMatchmaker algorithm and performs automated epitope virtual crossmatching at the initiation of the organ donation process. EpViX is a free, web-based application developed for use over the internet on a tablet, smartphone or computer. This program was developed using the Ruby programming language and the Ruby-on-Rails framework. To improve the user experience, the EpViX software interface was developed based on the best human–computer interface practices. To simplify epitope analysis and virtual crossmatching, the program was integrated with important available web-based resources, such as OPTN, IMGT/HLA and the International HLA Epitope Registry. We successfully developed a program that allows people to work collaboratively and effectively during the donation process by accurately predicting negative crossmatches, saving time and other resources.

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.

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.

OR53: HLA-EpiDB: A DATABASE FOR THE ANALYSIS OF HLA EPITOPE FREQUENCIES IN WORLDWIDE POPULATIONS

Aim The presence of anti-HLA donor-specific antibodies in transplant patients is a crucial factor related to tissue and graft rejection. These antibodies target specific regions of HLA proteins that are different from the transplant patient’s HLA proteins – termed HLA epitopes. Current efforts in matching for kidney transplantation often attempt to minimise the number of allele-level mismatches, and yet there is growing evidence that an HLA epitope-focussed approach for minimising mismatches may be more appropriate. In this study, we present the development of a new section within the Allele Frequency Net Database (AFND, www.allelefrequencies.net ), called HLA-EpiDB, for the storage of HLA epitope frequencies across worldwide populations. Methods To generate epitope frequencies we used seventeen genotype datasets and twenty-four haplotype datasets from 41 worldwide populations comprising more than 36,000 individuals, which were also stored in AFND. All populations datasets used in this study were typed at high resolution level for HLA-A, -B and -DRB1 loci, and nineteen also included HLA-C. To map the >2000 HLA Class I alleles that have been reported in IMGT/HLA database as of release 3.16.0 (April, 2014), we used the definition of HLA epitopes and mapping to HLA alleles established by the HLA Epitope Registry ( http://www.epregistry.ufpi.br/ ). Epitope frequencies (percentage of individuals carrying the epitope) were estimated using Hardy–Weinberg proportions for haplotype-derived data and by direct counting from genotype data. Results We created a new section in AFND called HLA-EpiDB, which allows users to filter for epitopes present in an individual HLA genotype, and analyse the likelihood of a population containing individuals with a closely matching epitope set. Another feature of HLA-EpiDB is that it permits users to display a cross-population comparison of epitope frequencies in a heatmap, or as a world map distribution for single epitopes. Conclusions We expect HLA-EpiDB to help transplant clinicians and the immunogenetics research community to better understand and determine HLA mismatch acceptability across worldwide populations. At present, we have estimated HLA epitope frequencies for HLA Class I alleles but Class II alleles will be added in the near future.

OR33: CLINICALLY USEFUL TOOL FOR COMPARING THEORETICAL AND EVIDENCE BASED HLA EPITOPES

Aim Theoretical HLA epitopes (THEps) were defined structurally by three-dimensional molecular modeling and amino acid sequence differences whereas evidence based HLA epitopes (EBHEps) considers the ability of a putative HLA epitope (HEps) to induce antibody. Such HEps are clinically relevant if the antibody is produced in humans on exposure to alloantigen. The aim of this study is to provide a cross reference tool for comparison of structurally defined HEps and EBHEps that may be of use to laboratories interested in defining patient sera by epitope reactivity. Method A comparative analysis of THEps from the International Registry of Antibody-Defined HLA Epitopes ( http://epregistry.com.br ), and evidence based Class I and Class II HLA epitopes from publications. The analysis was arranged in a cross referenced tabular format. Results We found 67 EBHEps that corresponded to theoretical Class I HEps epitope. Of the 67 theoretical Class I HEps, 7 were reactive with mouse monoclonal antibodies only and 60/67 epitopes were reactive with human allosera. Interestingly, 6 of the 60 human allosera - reactive epitopes were present on HLA-A and B, 2/60 were present on HLA-A, B and C, and 3/60 human reactive epitopes were present on HLA-B and C. We also found 55 EBHEps that corresponded to theoretical DR epitopes and16 evidence based DQ epitopes that corresponded to theoretical epitopes. Based on this data, tables were created to cross reference each of the THEps with the EBHEps thereby facilitating analysis of the patient’s antibody data. Conclusion The nomenclature of the theoretical epitopes ( http://epregistry.com.br ) is based on the position of the polymorphic amino acid residue in numerical order in the polypeptide chain followed by the single letter amino acid code (in alphabetical order). Evidence based epitopes had been published as a list in consecutive numerical order unrelated to the polymorphic amino acid position. One of the major goals of the HLA epitope registry is to identify clinically relevant HLA epitopes that have been verified experimentally with human allosera. The charts developed in our study allow for the convenient cross correlation between evidence based epitopes and theoretical epitopes that will be updated according to new evidence of human allosera reactivity. A.J. Norin: Speaker’s Bureau; Company/Organization; Immuncor - Lifecodes. Scientific/Medical Advisor; Company/Organization; ICON CL.

P124 : PREDICTION OF NEGATIVE FLOW CYTOMETRY CROSS MATCH BASED ON ALLELE/HLA EPITOPE ANALYSIS: A CASE REPORT

Patient C.A’s pre-transplant sera was found to be negative for antibodies against HLA Class I but strongly positive for antibodies against HLA Class II. The Class II specificities of the patient’s anti HLA antibodies were DR1, DR4, DR7 and DR10, DR17, DR18. The highest bead, DRB1∗01:02 was about10,000 MFI. In the single antigen bead kit used at our center, four DR4 beads were present: DRB1∗04:01, DRB1∗04:02, DRB1∗04:03, and DRB1∗04:05. Of the four, three were positive. The DRB1∗O4:02 bead was not reactive. In the past year, the patient was eligible for a deceased donor transplant. The deceased donor’s HLA type was HLA A2, A24, B7, B41, DR4, DR11, DQ7, DQ8. A flow cross match (FCXM) was performed. Though the donor’s HLA type was DR4, the B cell FCXM was negative, as well as the T cell FCXM. A possible explanation for the negative FCXM in the DR4 positive donor is that the donor’s HLA DR4 allele was DRB1∗04:02. In order for this explanation to be plausible, an epitope must be present on DRB1∗04:01, DRB1∗04:03, and DRB1∗04:05 (positive beads) but not on DRB1∗04:02 (negative bead). In real time, we searched the HLA epitope registry http://epregistry.ufpi.br/index/index for evidence of such an epitope. Epitope 70QA was present on the following Luminex alleles: DRB1∗01:01, DRB1∗01:02, DRB1∗04:01, DRB1∗04:03, DRB1∗04:04, DRB1∗04:05, DRB1∗14:02, DRB1∗15:01, DRB1∗15:02, DRB1∗15:03, DRB5∗02:02. We noted that this epitope excluded the DRB1∗04:02 allele. To further substantiate the reactivity of the patient’s sera, a FCXM was performed between the sera of the recipient against the B and T cells of a subject known to have the DRB1∗04:01 allele. The B cell FCXM was strongly positive, whereas the T cell FCXM was negative. It is a policy of our center not to transplant potential transplant patients with a positive B cell FCXM this particular case, the above referenced patient would be eligible to receive a kidney transplant from a donor with a DRB1∗04:02 type. Unfortunately, the current UNOS registry does not permit transplant centers to enter allele specific and/or epitope specific unacceptable antigen information. If the input of allele or epitope specific antigen data was initiated by UNOS, it would be extremely beneficial for transplant candidates who develop such allele specific antibodies. A.J. Norin: Speaker’s Bureau; Company/Organization; Immucor-Lifecodes. Scientific/Medical Advisor; Company/Organization; ICON CL.

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.