Marilyn Marrari

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

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

Detection of donor-specific HLA antibodies before and after removal of a rejected kidney transplant

UNLABELLED Serum analysis of patients considered for retransplantation has a potential limitation that the rejected allograft may absorb HLA antibodies. We have determined how the highly sensitive micro bead-based Luminex antibody-binding assay with single antigens can detect donor-specific HLA antibodies (DSA) in patients before and after surgical removal of a rejected allograft. This analysis was done for 65 allograft nephrectomy (allonx) cases contributed by 16 laboratories worldwide. In the HLA-A,B and -DRB1 mismatch categories the incidence of DSA reactivity pre-allonx and post-allonx was 64% vs 87% (p=0.0033) and 57% vs 86% (p=0.001), respectively. The frequencies of individual reactive antigens were also lower before allonx: for HLA-A,B antigens: 49% vs 75% (p<0.0001) and DRB1 antigens: 48% vs 79% (p=0.0001). On the other hand, no significant differences were seen between the pre-allonx and post-allonx frequencies of DSA to DRB3/4/5 (65% vs 78%, p=0.22) and DQ mismatches (76% vs 87%, p=0.18). CONCLUSION although the sensitive Luminex antibody assay can detect anti-donor antibodies in the presence of a rejected transplant, it is apparent that the antibody specificity pattern is often incomplete especially against the HLA-A, -B and DR mismatches. This understanding seems relevant to the determination of acceptable mismatches for patients considered for retransplantation.

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

Purpose of reviewThis review addresses the concept that human leukocyte antigen (HLA) antibody specificity should be determined to HLA epitopes rather than HLA antigens. Recent findingsHLAMatchmaker 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. SummaryA determination of the repertoire of immunogenic epitopes is important for HLA compatibility testing and the identification of acceptable mismatches for sensitized patients.

Multicenter evaluation of the flow cytometry T-cell crossmatch: results from the American Society of Histocompatibility and Immunogenetics-College of American Pathologists proficiency testing program.

BACKGROUND The performance characteristics and interlaboratory comparisons of the T-cell flow cytometry crossmatch remain largely unknown. METHODS This study was performed using data from the ASHI-CAP proficiency testing program. Four unknown sera and two unknown cells were sent to participating laboratories twice a year for 4 years. RESULTS In one survey in which different crossmatch techniques were compared, flow cytometry was slightly more sensitive than the antiglobulin method and considerably more sensitive than direct cytotoxicity. However, the proportion of participants in any given survey detecting antibodies in all sera expected to be positive was 50-60% and has not changed over the years. Failure to detect antibodies correlated with low antibody concentration, diluting the unknown serum by the testing laboratory, and with the instrument used. False positive results with normal sera were infrequent. Fluorescence intensity values were not standardized and were highly variable, but when fluorescence units reported by individual laboratories were divided by their own positive-negative cutoff values, results from different centers were more comparable. In general, fluorescence-to-cutoff ratios >5 correlated with complement binding activity, whereas values <5 denoted concentrations below those required to fix complement. CONCLUSIONS Flow cytometry, as used by most centers, is highly sensitive and allows relative antibody quantitation. Furthermore, the data define objective parameters that may help to standardize the test and improve its predictive value in clinical transplantation.

Detection of antibodies against HLA-C epitopes in patients with rejected kidney transplants.

BACKGROUND Although HLA-C matching is not considered in kidney transplantation, several reports have shown that anti-HLA-C antibodies are associated with rejection and graft failure. DNA-based typing methods can now accurately determine HLA-C compatibility and sensitive assays such as Luminex with single alleles can identify HLA-C antibodies. HLA-C displays considerable amino acid polymorphism that can be translated into a structurally defined epitope repertoire. METHODS We have analyzed post-allograft nephrectomy sera from 45 HLA-C mismatched cases submitted by 15 laboratories worldwide participating in the 15th International Histocompatibility Workshop. All of them had HLA class I antibodies detected by a Luminex-based solid phase method using single-allele beads. This study addressed the determination of antibodies against donor HLA-C mismatches. Analysis of antibody reactivity patterns was performed using HLAMatchmaker, a structurally based matching program that considers 56 HLA-C eplets to define antibody-reactive epitopes. Many eplets shared by groups of HLA-C antigens, whereas others are also shared with HLA-A and/or HLA-B antigens. RESULTS Twenty-seven patients (60%) had donor-specific HLA-C antibodies, significantly less than the donor-specific antibodies induced by HLA-A and HLA-B mismatches. HLA-C antibody responses correlated with the eplet loads of the HLA-C mismatches. There were 352 instances whereby a donor HLA-C eplet was mismatched and for 84 (24%) of them there was antibody reactivity with a particular eplet (69 instances) or an eplet pair (15 instances). The latter generally consisted of mismatched eplets paired with self-eplets shared between the immunizing HLA-C alleles and HLA alleles of the patient. Several HLA-C eplets exhibited a relatively high immunogenicity as evidenced by their frequencies of specific antibodies. CONCLUSION These findings demonstrate the importance of HLA-C mismatching in humoral sensitization and that HLA-C epitopes can induce specific antibodies. They illustrate the usefulness of HLAMatchmaker in understanding donor-specific antibody reactivity patterns and the determination of HLA mismatch acceptability when transplantation is considered.

Human Monoclonal Antibody Reactivity With Human Leukocyte Antigen Class I Epitopes Defined by Pairs of Mismatched Eplets and Self-Eplets

Aim. Humoral sensitization affects transplant outcome, and it is now apparent that human leukocyte antigen (HLA) antibodies are specific for epitopes rather than antigens. Such epitopes can be structurally defined by HLAMatchmaker, an algorithm that considers eplets as critical elements of epitopes recognized by alloantibodies. This study addressed the question how mismatched HLA antigens induce specific antibodies in context with eplet differences with the antibody producer. Methods. HLA class I-specific human monoclonal antibodies derived from women sensitized during pregnancy were tested in Luminex assays with single allele panels. Their epitope specificity was determined from reactivity patterns and eplet differences between immunizing antigen and the antibody producer. Results. This study focuses on the reactivity patterns of 10 monoclonal antibodies specific for epitopes defined by a mismatched eplet paired with a self-eplet shared between immunizing HLA antigens and HLA antigens of the antibody producer. The eplets in these pairs are between 7 and 16 Å apart, a sufficient distance for contact by two separate complementarity-determining regions of antibody. Conclusions. These findings demonstrate that immunizing antigens have mismatched eplets that can form antibody-reactive epitopes with self-configurations on the molecular surface. They seem to suggest that HLA antibodies can be produced by autoreactive B cells that have undergone receptor editing to accommodate the recognition of nonself-eplets, the driving force of the humoral alloresponse. This concept enhances our understanding of structural epitope immunogenicity and the interpretation of antibody reactivity patterns with HLA panels.

16th IHIW: a website for antibody-defined HLA epitope Registry.

The concept that HLA antibodies are specific for epitopes rather than HLA antigens is important not only for the determination of mismatch acceptability for sensitized patients but also for a better understanding of the antibody response to an HLA mismatch. Numerous publications describe epitope‐specific antibodies, but there is no standardized information about the repertoire of clinically relevant HLA epitopes. Under auspices of the 16th IHIW, we have developed a website‐based registry of antibody‐verified HLA epitopes. Epitope notations are based on HLA molecular modelling of amino acid residues in polymorphic sequence positions. Informative epitope‐specific antibodies had been induced by a transplant, transfusion or pregnancy and were monoclonal antibodies or eluates of sera absorbed with single HLA alleles. Antibody reactivity was determined in binding assays with single‐allele panels. Antibody producer/immunizer HLA types enhanced the characterization of specific epitopes. The Registry also includes epitopes described in original research publications. Based on the extent of antibody reactivity information, we assigned epitope status as confirmed (well documented) or provisional (more data are needed). At present, the Registry has 69 HLA‐ABC, 53 DRB1/3/4/5, 17 DQ, 8 DP and 22 MICA antibody‐verified epitopes and will be updated on a quarterly basis. Laboratories worldwide continue to submit data about previously unreported antibody‐specific epitopes. For each epitope, the website shows its amino acid composition and HLA alleles that share the epitope. Links show antibody reactivity patterns, sensitization information and references. Other links show molecular modelling of corresponding structural epitopes and polymorphic residue information for epitope‐carrying alleles. The website will also have a link to epitope frequency information in different populations. Search functions will list mismatched epitopes on mismatched alleles for selected HLA types. The HLA Epitope Registry will become a valuable resource for researchers interested in HLA compatibility at the epitope level and investigating antibody responses to HLA mismatches.

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.

Bone marrow transplantation for severe aplastic anemia using a phenotypically HLA-identical, SB-compatible unrelated donor.

A three-year-old boy with severe aplastic anemia (HLA-A1,B8(Bw6), Cw7,DR3, MB2, MT2, SB4/A1,B8 (Bw6), Cw7,DR3,MB2,MT2,SB-) received a bone marrow transplant from a phenotypically HLA-identical, SB-compatible female unrelated donor. This donor was selected from eighteen HLA-A1,-B8,-blood donors after extended serotyping, mixed leukocyte culture testing and secondary proliferation assays with primed lymphocyte typing reagents specific for SB. Although patient cells proliferated well as responders in MLR, their stimulatory capability was greatly impaired. Because the patient had inherited the same serological HLA-D haplotype from each parent, it was concluded that a compatible unrelated donor must be homozygous for the same HLA-D antigens as the patient. This HLA-D homozygosity was demonstrated by the lack of MLR responses of both parents to stimulators from the donor. The SB typing results suggested SB compatibility because both the patient and the donor typed as SB4,-. Following bone marrow transplantation, there was rapid hematopoietic engraftment. The patient developed severe diarrhea caused by graft-versus-host disease of the gastrointestinal tract, which necessitated hyperalimentation. He is currently eighteen months posttransplant with full hematopoietic reconstitution and moderate chronic skin graft-versus-host disease.

Determination of HLA-A,B residue mismatch acceptability for kidneys transplanted into highly sensitized patients: a report of a collaborative study conducted during the 12th International Histocompatibility Workshop.

BACKGROUND During the 12th International Histocompatibility Workshop, a collaborative study between 35 laboratories was conducted on a group of highly allosensitized patients who had received a kidney transplant from 1981 to 1995. The major goal of the study was to assess how serum screening against a large cell panel could determine donor HLA mismatch acceptability in relation to graft outcome. METHODS Twenty laboratories participated in an extensive screening of 92 high panel-reactive antibody (PRA) sera from patients at 29 transplant centers worldwide; each patient had received a kidney allograft from an HLA-A,B mismatched unrelated donor. Screening was done by complement-dependent lymphocytotoxicity and antihuman globulin augmentation techniques using a common protocol and shared standardized reagents. After an extensive quality-control assessment, we selected data from 14 participants who had screened the sera against a combined panel of 535 HLA-typed cells. RESULTS With the 2x2 table-based Multiscreen computer program, we could readily determine for virtually every patient the significant correlations between serum reactivity and the presence of panel cell markers, including private and public HLA-A,B epitopes and amino acid residues assigned from published sequencing data. Donor mismatch acceptability was assessed at the amino acid residue level. In the complement-dependent lymphocytotoxicity (n=49; PRA=84.1+/-12.1%) and antihuman globulin (n=60; PRA=92.5+/-5.8%) groups, the 3-month graft survivals were 28% and 30% lower for unacceptable residue mismatches. CONCLUSIONS These studies underscore the importance of a comprehensive serum screening analysis in the selection of appropriately mismatched donors for highly sensitized transplant patients.