Adaptation of HLA testing to characterize the cynomolgus macaque MHC polymorphisms and alloantibody signatures.

Nonhuman primates are the closest animal models to humans with respect to genetics and physiology. Consequently, a critical component of immunogenetics research relies on drawing inferences from the cynomolgus macaque to inform human trials. Despite the conserved organization of the Major Histocompatibility Complex (MHC) between cynomolgus macaques and humans, MHC genotyping of cynomolgus macaques is challenging due to high rates of copy number variants, duplications, and rearrangements, particularly at the MHC class I loci. Furthermore, the limited availability of commercial reagents specific to cynomolgus macaques that can be used to characterize anti-MHC class I and class II antibody (Ab) specificities in cynomolgus macaques presents a major bottleneck in translational research. Here we successfully characterized cynomolgus macaque Mafa class I and class II serologic specificities in 86 animals originating from various geographical regions using the complement dependent cytotoxicity (CDC) assay with human HLA class I and class II monoclonal antibody (mAb) typing trays. Further, we successfully induced and characterized anti-Mafa class I and class II alloantibody specificity using HLA single antigen bead assays. We also subsequently tracked the alloAb burden in the animals during treatment with anti-B lymphocyte stimulator (BLyS) treatment. Altogether, these methods can be easily used in translational research to serotype MHC class I and class II specificity in macaques, characterize their alloAb specificity, and evaluate the efficacy of novel therapeutic modalities in depleting circulating alloAbs in these animals.

Autobiographical perspectives on HLA epitopes: Past, present and future.

This article describes my personal perspectives on HLA epitopes. It is not intended to be a general review of HLA epitopes as several versions have been published before. This article is an autobiographical reflection with three sections. The first part named "Past" is intended to show how traditional HLA typing and serological HLA data might be interpreted with epitopes. The second section named "Present" describes my experience with HLA epitopes including antibody verification and the concept of ^eplet load. The third part, "Future", expresses my (thoughts about HLA epitopes and their application in the clinical setting. The list of cited References includes publications selected from the HLAMatchmaker website www.epitopes.net. Most of these references have PDF documents than can be downloaded without charge.

Immunogenetics of heteroclitic recognition of HLA-DQB1 55R eplet specificity by human alloantibody.

Heteroclitic antibodies bind to a related antigen with higher affinity than to the immunizing antigen to which they were generated. This uncommon phenomenon is not well characterized for antibodies to HLA antigens. Here we analyzed allosera reactivity from two transplant recipients sensitized to mismatched donor alleles DQB1*06:01 and DQB1*06:02 respectively. Epitope analysis demonstrated the reactivity of both sera was restricted to DQB1*04, 05, and 06 alleles, with a specificity associated with the 55R eplet. Serum from one of these subjects (TE) was significantly more reactive with DQB1*04 alleles than the immunizing DQB1*06:01 or other alleles, a pattern not present in serum from the other patient. Antibody absorption/elution experiments using B cell lines expressing DQB1*06:01 or DQB1*04:02 alleles confirmed that the heteroclitic TE antibody eluted from cells carrying DQB1*06:01 was significantly more reactive with beads carrying the DQB1*04 alleles than with the DQB1*06 or other alleles. The significantly higher reactivity of the heteroclitic alloantibody with DQB1*04 specificity was explained structurally by variations of amino acid residues within 3.5 Å of 55R. These findings have important implications for the interpretation of DQ alloantibody cross-reactivity frequently observed in transplant recipients.

Epitope-based human leukocyte antigen matching for transplantation: a personal perspective of its future.

PURPOSE OF REVIEW: This study reflects my personal experience with the characterization of human leukocyte antigen (HLA) epitopes and their significance in HLA matching for transplantation. It offers a subjective assessment what further studies are needed to have this concept be applied in the clinical setting. RECENT FINDINGS: This study addresses the structural characteristics of antibody-reactive HLA epitopes determined by different methods, eplet-associated antibody analysis and acceptable mismatching for sensitized patients and eplet immunogenicity and determination of mismatch permissibility. BASIC IMPLICATIONS: for clinical practice and research consider the need for further studies of the structural basis of antibody-verified HLA epitopes determined in different techniques and their clinical relevance, the biological basis of epitope immunogenicity and determinations of permissible mismatches and a computerized clinical transplant database with an Artificial Intelligence component that can generate evidence-based information for the practical application of epitope-based HLA matching.

Case report: A transplant candidate with unexpected serum reactivity against the 45KE eplet on HLA-B alleles.

This case report describes the serum antibody specificity against the 45KE eplet which had not been yet shown to be antibody-verified. This antibody was produced by a 41-year-old European male with Berger's disease. His serum had HLA class I antibody reactivity as determined in IgG binding assays with single allele panels (OneLambda, ThermoFisher, Lot 8 and Lot 9). The HLAMatchmaker analysis revealed reproducible serum reactivity only with alleles carrying the 45KE eplet. The cause of this 45KE-specific immunization is unknown because this male patient had never been transfused nor received a previous transplant, Moreover, his mother's HLA type did not have any 45KE-carrying allele. This finding might be related to observations reported in the literature about the appearance of HLA-reactive antibodies following influenza vaccination but this possibility could not be investigated.

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.

Are We Ready for Epitope-Based HLA Matching in Clinical Organ Transplantation?

This overview describes recent developments demonstrating the significance of epitopes in HLA antibody responses and matching for organ transplantation. HLA epitopes are defined by molecular modeling and amino acid comparisons between HLA alleles and the HLAMatchmaker algorithm considers eplets as essential components. Each allele represents a distinct string of eplets and matching is done by aligning donor and recipient strings. Evidence is summarized how mismatched eplet loads affect antibody responses and transplant outcomes. Epitope-based matching has been applied not only to identify acceptable mismatches for sensitized transplant candidates but also to identify more suitably mismatched donors for nonsensitized patients. Three recently proposed theories will further our understanding of the immunogenicity of individual HLA eplets.It has become apparent that epitope-based matching is superior to antigen matching; we should be ready soon to apply this principle in the clinical transplant setting very soon.

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.

Reflections on HLA Epitope-Based Matching for Transplantation.

HLA antibodies are primary causes of transplant rejection; they recognize epitopes that can be structurally defined by eplets. There are many reviews about HLA epitope-based matching in transplantation. This article describes some personal reflections about epitopes including a historical perspective of HLA typing at the antigen and allele levels, the repertoires of antibody-verified HLA epitopes, the use of HLAMatchmaker in determining the specificities of antibodies tested in different assays, and, finally, possible strategies to control HLA antibody responses.

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.

Epitope analysis of DQ6-reactive antibodies in sera from a DQ6-positive transplant candidate sensitized during pregnancy.

This case report describes DQ6-reactive serum antibody reactivity in a patient who types as DQ6. DNA typing showed DQB1*06:09 on the antibody producer and serum reactivity with DQB1*06:01, *06:02 and *06:03 but not with *06:04 and *06:09. HLAMatchmaker serum analysis showed antibody reactivity with a new antibody-verified 85VA eplet on DQB but additional reactivity with DQB1*02:01 could not be readily interpreted. After applying the nonself-self algorithm of HLA immunogenicity we have identified a new DQB epitope structurally described as 140A2+130R+135D and shared by DQB1*02:01 and DQB1*05:01 and DQB1*06:02 of the immunizer.

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.

Usefulness of the Nonself-Self Algorithm of HLA Epitope Immunogenicity in the Specificity Analysis of Monospecific Antibodies Induced during Pregnancy.

BACKGROUND: HLAMatchmaker is a program to analyze the epitope specificities of HLA antibodies. It considers each HLA allele as a string of eplets. Intralocus and interlocus comparisons between donor and recipient alleles offer a structural assessment of compatibility and an analysis of allele panel reactivity patterns can generate information about epitope specificities of HLA antibodies. However, HLAMatchmaker cannot always generate conclusive interpretations of reactivity patterns of all monospecific antibodies, which by definition recognize single epitopes. HYPOTHESIS: We have therefore developed a new antibody analysis approach that utilizes the nonself-self algorithm of HLA epitope immunogenicity. It is based on the concept that HLA antibodies originate from B-cells with immunoglobulin receptors to self-HLA epitopes on one given allele and which can be activated by epitopes defined by a few nonself residue differences whereas the remainder of the structural epitope of the immunizing allele consists of self residues. METHODS: Three human monoclonal class I antibodies from HLA typed women sensitized during pregnancy were tested in Ig-binding assays with single alleles on a Luminex platform. FINDINGS: Three new HLA epitopes were identified; they are defined by combinations of nonself- and self-residues for one allele of the antibody producer. CONCLUSION: The nonself-self paradigm of HLA epitope immunogenicity offers a second approach to analyze HLA antibody specificities.

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 leukocyte antigen epitope antigenicity and immunogenicity.

PURPOSE OF REVIEW: Human leukocyte antigen (HLA) antibodies are now recognized as being specific for epitopes which can be defined structurally with amino acid differences between HLA alleles. This article addresses two general perspectives of HLA epitopes namely antigenicity, that is their reactivity with antibody and immunogenicity, that is their ability of eliciting an antibody response. RECENT FINDINGS: Single-antigen bead assays have shown that HLA antibodies recognize epitopes that are equivalent to eplets or corresponding to eplets paired with other residue configurations. There is now a website-based Registry of Antibody-Defined HLA Epitopes (http://www.epregistry.com.br). Residue differences within eplet-defined structural epitopes may also explain technique-dependent variations in antibody reactivity determined in Ig-binding, C1q-binding and lymphocytotoxicity assays.HLA antibody responses correlate with the numbers of eplets on mismatched HLA antigens, and the recently proposed nonself-self paradigm of epitope immunogenicity may explain the production of epitope-specific antibodies. SUMMARY: These findings support the usefulness of HLA matching at the epitope level, including the identification of acceptable mismatches for sensitized patients and permissible mismatching for nonsensitized patients aimed to reduce HLA antibody responses.

Early acute antibody-mediated rejection of a negative flow crossmatch 3rd kidney transplant with exclusive disparity at HLA-DP.

Donor-specific alloantibodies (DSA) to HLA-DP may cause antibody-mediated rejection (AMR), especially in re-transplants. We describe the immunization history of a patient who received 3 kidney transplants; the 3rd kidney was completely matched except at DPA1 and DPB1. Prior to the 3rd transplant, single antigen bead analysis (SAB) showed DSA reactivity against DPA1 shared by the 1st and 3rd donors, but B and T flow crossmatch (FXM) results were negative. Within 11 days the 3rd transplant underwent acute C4d+ AMR which coincided with the presence of complement (C1q)-binding IgG1 DSA against donor DPA1 and DPB1. Using HLAMatchmaker and SAB, we provide evidence that eplet (epitope) spreading on DPA1 and eplet sharing on differing DPB1 alleles of the 1st and 3rd transplants was associated with AMR. Since weak DSA to DPA1/DPB1 may induce acute AMR with negative FXM, donor DPA1/DPB1 high resolution typing should be considered in sensitized patients with DP-directed DSA.

HLA epitope based matching for transplantation.

As important risk factors for transplant rejection and failure, HLA antibodies are now recognized as being specific for epitopes which can be defined structurally with amino acid differences between HLA alleles. Donor-recipient compatibility should therefore be assessed at the epitope rather than the antigen level. HLAMatchmaker is a computer algorithm that considers each HLA antigen as a series of small configurations of polymorphic residues referred to as eplets as essential components of HLA epitopes. It includes epitopes on antigens encoded by all HLA-A, B, C, DR, DQ and DP loci as well as MICA. HLA epitopes have two characteristics namely antigenicity, i.e. the reactivity with antibody and immunogenicity, i.e. the ability of eliciting an antibody response. This article addresses the relevance of determining epitope-specificities of HLA antibodies, the effect of epitope structure on technique-dependent antibody reactivity and the identification of acceptable mismatches for sensitized patients considered for transplantation. Permissible mismatching for non-sensitized patients aimed to prevent or reduce HLA antibody responses could consider epitope loads of mismatched antigens and the recently developed nonself-self paradigm of epitope immunogenicity.

Structural aspects of HLA class I epitopes reacting with human monoclonal antibodies in Ig-binding, C1q-binding and lymphocytotoxicity assays.

This study addresses the reactivity patterns of human cytotoxic HLA class I epitope-specific monoclonal antibodies in Ig-binding and complement component C1q-binding Luminex assays in comparison with complement-dependent lymphocytotoxicity data reported at the 13th International HLA Workshop. Some monoclonal antibodies reacted similarly with epitope-carrying alleles in all three assays but others showed different reactivity patterns. These reactivity differences were analyzed with HLAMatchmaker and we incorporated the concept that eplets are essential parts of structural epitopes which can contact the six Complementarity Determining Regions (CDRs) of antibody. The data show that technique-dependent reactivity patterns are associated with distinct differences between polymorphic amino acid configurations on eplet-defined structural epitopes. The findings have been viewed in context of antigen-antibody complex formation that results in the release of free energy necessary to stabilize binding and to induce conformational changes in the antibody molecule to expose the C1q binding site, the first step of complement activation. Moreover the amount of free energy should be sufficient to induce a conformational change of C1q thereby initiating the first stages of the classical complement cascade leading to lymphocytotoxicity. The complement-fixing properties of HLA antibodies require not only specific recognition of eplets but also depend on interactions of other CDRs with critical amino acid configurations within the structural epitope. Eplet-carrying alleles that lack such configurations may only bind with antibody. This concept is important to our understanding whether or not complement-fixing donor-specific HLA antibodies can initiate antibody-mediated rejection.