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.

Utility of cross-matched platelet transfusions in patients with hypoproliferative thrombocytopenia: a systematic review.

BACKGROUND: Multiply transfused hypoproliferative thrombocytopenic (HT) patients with alloimmune transfusion refractoriness require specially selected platelets (PLTs). Cross-matching apheresis PLTs is a popular support option, avoiding requirements for large panels of typed donors for HLA-based selection. We undertook a systematic review of the utility of various cross-matching techniques on mortality reduction, prevention of hemorrhage, alloimmunization and refractoriness, and improvement in PLT utilization or count increments. STUDY DESIGN AND METHODS: A systematic review to December 2012 was conducted of MEDLINE, EMBASE, and Cochrane databases along with a bibliographic search of pertinent references. RESULTS: Of 146 retrieved citations, 20 met inclusion criteria. Eleven more were chosen from bibliographies, describing 29 unique cohorts. All but five enrolled transfusion-refractory, predominantly alloimmunized patients. Cross-match impact on mortality and hemorrhage could not be assessed from these studies. Two studies demonstrated durable corrected count increments and/or breadth of alloimmunization throughout cross-match support; none addressed development or persistence of refractoriness. In alloimmunized refractory patients and nonrefractory cohorts with greater than 25% alloimmunization, higher increments were seen with cross-match-compatible PLTs than incompatible or un-cross-matched units. In two nonrefractory, nonalloimmunized cohorts, the lack of utility of cross-match was reflected by test sensitivity of less than 20%. Comparison of cross-matched PLT success with that of HLA-identical units revealed inferior success rates for the former in one study and equivalent rates in another. No trend was observed regarding relative utility of the various commonly employed techniques. CONCLUSION: Cross-matched PLTs are useful in increasing PLT counts in alloimmunized, transfusion-refractory HT patients, but data about their impact on hemorrhage and mortality are lacking.

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.

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.

Efficacy of HLA-matched platelet transfusions for patients with hypoproliferative thrombocytopenia: a systematic review.

BACKGROUND: HLA-matched platelets (PLTs) are widely used to transfuse patients but the effectiveness of HLA matching has not been well defined and the cost is approximately five times the cost of preparing the random-donor PLTs. The objective of this systematic review was to determine whether HLA-matched PLTs lead to a reduction in mortality; reduction in frequency or severity of hemorrhage; reduction in HLA alloimmunization, refractoriness, or PLT utilization; or improvement in PLT count increment in patients with hypoproliferative thrombocytopenia. STUDY DESIGN AND METHODS: We conducted a literature search of MEDLINE, Cochrane Controlled Register of Clinical Trials, EMBASE, and PubMed databases to April 2012. RESULTS: A total of 788 citations were reviewed and 30 reports were included in the analysis. Most studies did not include technologies currently in use for HLA typing or detection of HLA antibodies as 75% were conducted before the year 2000. None of the studies were adequately powered to detect an effect on mortality or hemorrhage. HLA-matched PLTs did not reduce alloimmunization and refractoriness rates beyond that offered by leukoreduction, and utilization was not consistently improved. HLA-matched PLTs led to better 1-hour posttransfusion count increments and percentage of PLT recovery in refractory patients; however, the effect at 24 hours was inconsistent. CONCLUSION: The correlation of the PLT increment with other clinical outcomes and the effect of leukoreduction on HLA-matched PLT transfusion could not be determined. Prospective studies utilizing current technology and examining clinical outcomes are necessary to demonstrate the effectiveness of HLA-matched PLT transfusion.

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.

Clinical usefulness of HLAMatchmaker in HLA epitope matching for organ transplantation.

HLAMatchmaker is a computer algorithm that determines HLA compatibility at the structural level. Donor-recipient histocompatibility is assessed with polymorphic amino acid configurations that represent structurally defined elements of HLA epitopes originally assigned as triplets and more recently as eplets. For many patients, HLAMatchmaker can identify mismatched HLA antigens that can be considered compatible at the structural level. Structurally based HLA matching reduces humoral allosensitization and correlates with good transplant outcome. Moreover, HLAMatchmaker is useful in the analysis of serum antibody reactivity and benefits the strategy of identifying acceptable mismatches for highly sensitized patients.

High-throughput sequencing defines donor and recipient HLA B-cell epitope frequencies for prospective matching in transplantation.

Compatibility for human leukocyte antigen (HLA) genes between transplant donors and recipients improves graft survival but prospective matching is rarely performed due to the vast heterogeneity of this gene complex. To reduce complexity, we have combined next-generation sequencing and in silico mapping to determine transplant population frequencies and matching probabilities of 150 antibody-binding eplets across all 11 classical HLA genes in 2000 ethnically heterogeneous renal patients and donors. We show that eplets are more common and uniformly distributed between donors and recipients than the respective HLA isoforms. Simulations of targeted eplet matching shows that a high degree of overall compatibility, and perfect identity at the clinically important HLA class II loci, can be obtained within a patient waiting list of approximately 250 subjects. Internal epitope-based allocation is thus feasible for most major renal transplant programs, while regional or national sharing may be required for other solid organs.

Long-term outcome of human leukocyte antigen mismatching in liver transplantation: results of the National Institute of Diabetes and Digestive and Kidney Diseases Liver Transplantation Database.

UNLABELLED: A perfect or nearly perfect human leukocyte antigen (HLA) match has been associated with better immediate and long-term survival of diseased donor kidney transplants. However, the effect of HLA matching for hepatic allografts remains poorly defined. Using data from the National Institutes of Diabetes and Digestive and Kidney Diseases Liver Transplantation Database, we investigated the association between HLA mismatches and hepatic allograft survival, disease recurrence, and immunosuppression interactions. A, B, and DR loci were used to calculate total mismatch scores of 0 (no mismatches in any loci) to 6 (mismatches in all loci). Seven hundred ninety-nine adults (male, 55%; female, 45%) underwent 883 liver transplants. The 10-year graft survival according to total mismatch score was as follows: 0-2, 60%; 3-4, 54%; and 5-6, 57%. There was a negative effect of mismatching at the A locus on patient survival, with shorter survival for patients with 1 or 2 mismatches compared with 0 mismatches [P = 0.05, hazard ratio (HR) = 1.6]. Patients on tacrolimus with 1 or 2 mismatches at B or DR loci appeared to have increased rates of patient and graft survival compared to patients with 0 mismatches, with the appearance of a protective effect of tacrolimus (HR = 0.67). The effect of HLA mismatching was more pronounced on certain disease recurrences. DR-locus mismatch increased recurrence of autoimmune hepatitis (P = 0.01, HR = 4.2) and primary biliary cirrhosis (P = 0.04, HR = 2). Mismatch in the A locus was associated with more recurrence of hepatitis C virus (P = 0.01, HR = 1.6) and primary sclerosing cholangitis (P = 0.03, HR = 2.9). CONCLUSION: Mismatching at the A locus decreases patient survival in liver transplant recipients, and mismatching at the DR and A loci affects recurrence of autoimmune liver diseases and hepatitis C, respectively.

HLAMatchmaker-based definition of structural human leukocyte antigen epitopes detected by alloantibodies.

PURPOSE OF REVIEW: This review addresses the concept that human leukocyte antigen (HLA) antibody specificity should be determined to HLA epitopes rather than HLA antigens. RECENT FINDINGS: HLAMatchmaker is a computer algorithm that considers small configurations of polymorphic residues referred to as eplets as essential components of HLA epitopes. This overview describes recent developments that have increased our understanding of structural epitope antigenicity, that is, reactivity with specific antibody and immunogenicity, that is, its ability to induce an antibody response. SUMMARY: A determination of the repertoire of immunogenic epitopes is important for HLA compatibility testing and the identification of acceptable mismatches for sensitized patients.

HLAMatchmaker-defined triplet matching is not associated with better survival rates of patients with class I HLA allele mismatched hematopoietic cell transplants from unrelated donors.

This report addresses the concept that permissible HLA mismatching, that is, mismatches that do not generate an allogeneic response, in hematopoietic stem cell transplantation (HCT) can be determined with structural similarity of polymorphic regions. We have applied the triplet version of a structural algorithm called HLAMatchmaker, which considers short sequences involving polymorphic amino acid residues on the molecular surface as key elements of immunogenic epitopes. The triplet matching effect was analyzed in a National Marrow Donor Program dataset consisting of 744 unrelated hematopoietic cell transplantation cases with 1 HLA-A, -B, or -C mismatch and 1690 fully HLA-A, -B, -C, -DR, or -DQ allele matched cases. In multivariate models adjusting for other significant clinical risk factors, the degree of triplet mismatching did not significantly correlate with patient survival, engraftment, or acute graft-versus-host disease (aGVHD). Other structurally based strategies should be pursued to identify permissible HLA mismatches in HCT.

Extending options for highly sensitized patients to receive a suitable kidney graft.

Highly sensitized patients (anti-HLA) on the kidney waiting list wait longer for a suitable crossmatch negative organ. At the moment there are two strategies to enhance transplantation of these patients. One approach is the determination of acceptable HLA mismatches and application of this knowledge for the selection of crossmatch negative donors, and the second is the desensitization of patients with intravenous immunoglobulin-based protocols to enable transplantation of an organ from a donor towards which antibodies were originally present. Both approaches have advantages and disadvantages and are only successful in a proportion of the patients. The optimal solution is an integrated strategy whereby desensitization is used for those patients for whom the acceptable mismatch approach is not successful.

Structurally based epitope analysis of major histocompatibility complex class I-related chain A (MICA) antibody specificity patterns.

Recent studies have suggested a clinical significance to the detection of anti-major histocompatibility complex class I-related chain A (MICA) antibodies in transplantation. We have developed an eplet-based version of the HLAMatchmaker algorithm to assess the epitope specificity of these antibodies. Molecular viewing of the MICA structure and the determination of amino acid sequence differences between MICA alleles has yielded a repertoire of 38 potentially immunogenic MICA eplets. These eplets are based on the functional epitope structure that considers a configuration of amino acids within a 3-Angstrom radius of an antibody-accessible polymorphic residue on the molecular surface. In this study MICA-reactive sera were screened in Luminex assays with single MICA allele panels and analyzed with HLAMatchmaker. We identified reactivity patterns that correspond to eplet-specific antibodies to identify of unacceptable MICA mismatches including those alleles that have not been tested with the serum. In conclusion, HLAMatchmaker is a useful algorithm to analyze the reactivity patterns of anti-MICA antibodies and the determination of MICA mismatch acceptability at the structural level.

Retransplant candidates have donor-specific antibodies that react with structurally defined HLA-DR,DQ,DP epitopes.

This report describes a detailed analysis how donor-specific HLA class II epitope mismatching affects antibody reactivity patterns in 75 solid organ transplant recipients with an in situ allograft and who were considered for retransplantation. Sera were tested for antibodies in a sensitive antigen-binding assay (Luminex) with single class II alleles. Their reactivity was analyzed with HLAMatchmaker, a structural matching algorithm that considers so-called eplets to define epitopes recognized by antibodies. Only 24% of the patients showed donor-specific anti-DRB1 antibodies and there was a significant correlation with a low number of mismatched DRB1 eplets. This low detection rate of anti-DRB1 antibodies may also be due to allograft absorption. In contrast, antibodies to DRB3/4/5 mismatches were more common. Especially, 83% of the DRB4 (DR53) mismatches resulted in detectable antibodies against an eplet uniquely found on DR53 antigens. Donor-specific DQB mismatches led to detectable anti-DQB antibodies with a frequency of 87%. Their specificity correlated with eplets uniquely found on DQ1-4. The incidence of antibodies induced by 2-digit DQA mismatches was 64% and several eplets appeared to play a dominant role. These findings suggest that both alpha and beta chains of HLA-DQ heterodimers have immunogenic epitopes that can elicit specific antibodies. About one-third of the sera had anti-DP antibodies; they reacted primarily with two DPB eplets and an allelic pair of DPA eplets. These data demonstrate that HLA class II reactive sera display distinct specificity patterns associated with structurally defined epitopes on different HLA-D alleles.

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.

HLAMatchmaker: a molecularly based algorithm for histocompatibility determination. V. Eplet matching for HLA-DR, HLA-DQ, and HLA-DP.

This report describes the design of the eplet version of HLAMatchmaker to determine class II compatibility at the structural level. This matching algorithm is based on the hypothesis, developed from molecular modeling of crystallized antigen-antibody complexes, that functional epitopes are represented by patches of surface-exposed nonself-amino acid residues surrounded by residues within a 3-A radius. Patch determinations with a molecular viewer of crystalline structural models downloaded from the Entrez Molecular Modeling Database Web site led to the identification of 44 DRB, 33DQB, 29 DQA, 20 DPB, and 9 DPA unique combinations of polymorphic positions. The residue compositions of these patches were then determined from amino acid sequences. This analysis resulted in a repertoire of 146 DRB, 74 DQB, 58 DQA, 45 DPB, and 19 DPA eplets. In many eplets, the residues are in short linear sequences, but many other eplets have discontinuous sequences of residues that cluster on or near the molecular surface. This analysis has also shown that all serologically defined DR and DQ antigens detectable by monospecific antibodies have unique eplets. Other eplets are present in groups of class II antigens, many of which appear as cross-reacting. The eplet version of HLAMatchmaker should be considered as a hypothetical model for the structural assessment of donor-recipient compatibility and the determination of mismatch acceptability for sensitized patients. This computer algorithm can be downloaded from the HLA Matchmaker Webside at http://tpis.upmc.edu.