Public HLA Research Articles

High-Definition Genomic Analysis of HLA Genes Via Comprehensive HLA Allele Genotyping.

HLA is essential for various medical applications, such as genomic studies of multifactorial diseases, including immune system and inflammation-related disorders. Therefore, an accurate HLA typing method that is applicable for any allele registered in HLA allele databases is required to deduce scientific evidence related to disorders. Here, we describe a method for determining HLA alleles from next-generation sequencing (NGS) results by using currently available HLA sequence data in public HLA databases and show its application in association analysis.

Deciphering HLA-I motifs across HLA peptidomes improves neo-antigen predictions and identifies allostery regulating HLA specificity.

The precise identification of Human Leukocyte Antigen class I (HLA-I) binding motifs plays a central role in our ability to understand and predict (neo-)antigen presentation in infectious diseases and cancer. Here, by exploiting co-occurrence of HLA-I alleles across ten newly generated as well as forty public HLA peptidomics datasets comprising more than 115,000 unique peptides, we show that we can rapidly and accurately identify many HLA-I binding motifs and map them to their corresponding alleles without any a priori knowledge of HLA-I binding specificity. Our approach recapitulates and refines known motifs for 43 of the most frequent alleles, uncovers new motifs for 9 alleles that up to now had less than five known ligands and provides a scalable framework to incorporate additional HLA peptidomics studies in the future. The refined motifs improve neo-antigen and cancer testis antigen predictions, indicating that unbiased HLA peptidomics data are ideal for in silico predictions of neo-antigens from tumor exome sequencing data. The new motifs further reveal distant modulation of the binding specificity at P2 for some HLA-I alleles by residues in the HLA-I binding site but outside of the B-pocket and we unravel the underlying mechanisms by protein structure analysis, mutagenesis and in vitro binding assays.

The beneficial role of inhibitory KIR genes of HLA class I NK epitopes in haploidentically mismatched stem cell allografts may be masked by residual donor-alloreactive T cells causing GVHD.

HLA allele mismatches will provoke T-cell alloreactivity after allogeneic stem cell transplantation. As donors and recipients are usually HLA matched, the public HLA epitopes that are recognized by natural killer (NK) cells (NK epitopes) are rarely mismatched, and therefore there is rarely potential for NK alloreactivity arising from the absence of ligands for inhibitory killer immunoglobulin-like receptors (KIR). Transplants using related donors sharing only one haplotype (haploidentical donors) represent a setting in which NK epitopes are often mismatched, thus resulting in the potential for NK alloreactivity. We have analyzed engraftment, acute graft vs host disease (GVHD), leukemia relapse, and survival in 62 haploidentical transplants in relationship with potential NK alloreactivity, inhibitory, and activating KIR genes of class I HLA NK epitopes. Potential NK alloreactivity in the rejection direction was not associated with any outcome variable. Potential NK alloreactivity in the GVHD direction was associated with an increased incidence of severe GVHD and poorer patient survival but not with non-engraftment nor leukemia relapse. A higher number of activating KIR receptors in the genome of the donor was associated with a higher prevalence of GVHD. These results suggest that lack of extensive T-cell depletion in haploidentical transplantation is associated with high GVHD rates and diminishes the benefits of NK-cell alloreactivity.

Antibodies to private and public HLA class I epitopes in platelet recipients.

Transfusions or pregnancies can cause immunization against private HLA determinants and public epitopes shared by more than one private HLA antigen. HLA antibodies are correlated with febrile transfusion reactions, lower platelet response following platelet transfusion, and an increased rate of renal transplant rejection. Until now, antibody specificities in alloantisera from platelet recipients have been poorly characterized. Consecutive serum screens from platelet recipients were analyzed for antibodies against private HLA class I antigens and public HLA epitopes using a serum analysis program based on the 2 x 2 table analysis of correlations. Serum screens of highly immunized patients and of patients with new alloimmunization events were reviewed separately. Of the serum screens from 566 platelet recipients, 1577 indicated alloimmunization (panel-reactive antibodies >5%). The program assigned a specificity in 1024 of these screens (64.9%) and at least once in 522 of 566 patients (92.2%). In 267 patients, antibodies detecting public epitopes in the combined A- or B-locus cross-reacting groups were found; other public markers were detected in 39 patients. Patterns of reactivity were remarkably less stable than in patient groups previously studied. In many patients, antibodies with apparent private epitope specificity preceded the identification of antibodies against a shared marker of the same cross-reactive group. However, the disappearance of antibodies (whether or not this was followed by a new antibody against a private or public marker belonging to another cross-reacting group) was also observed. The computerized analysis of microlymphocytotoxicity tests enhances the rate of antibody specification in sera from platelet recipients with lymphocytotoxic antibodies. The identified antibodies should be taken into account in the selection of platelet donors. The data confirm and extend previous observations on HLA class I antibodies and elucidate new alloimmunization events.

The humoral immune response against an HLA class I allodeterminant correlates with the HLA-DR phenotype of the responder.

The genetic basis for control of alloantibody responses against foreign HLA histocompatibility antigens has never been delineated. The most likely postulate would be that HLA class II alloantigens of the host regulate the response through their ability to present processed HLA allopeptide fragments for the cognate interaction between CD4+ T lymphocytes and B lymphocytes that leads to IgG antibody synthesis. We have analyzed our allosensitized transplant patient population with regard to humoral responsiveness to a serologically defined public HLA class I epitope, Bw4. Peptides representing the linear sequence of the Bw4 epitope (amino acids 74-86) and the alternative Bw6 epitope were synthesized and assayed for binding to a panel of HLA homozygous lymphoblastoid B cells using a quantitative fluorescence binding assay. We found that 73% of patients who have produced a HLA-Bw4-specific alloantibody express either the HLA-DRB1*01 or HLA-DRB1*03 alloantigen; 19% of the remaining responders expressed HLA-DRB1*04. Analysis of the United Network for Organ Sharing Transplant Registry indicated that the survival of cadaver renal allografts mismatched for Bw4 was significantly compromised in sensitized DRB1*01+ or DRB1*03+ recipients (P<0.01). In vitro, the Bw4 peptide bound strongly to DRB1*01+ and DRB1*03+ lymphoblastoid B cells; no similar binding was observed with Bw6 peptide. These findings were confirmed using murine fibroblast lines transfected with HLA-DR alpha/beta genes and by solid-phase enzyme-linked immunosorbent assay using purified HLA-DR alloantigen. We conclude that there are at least two human Ir genes, HLA-DRB1*01 and HLA-DRB1*03, that confer a high risk for both humoral allosensitization and renal allograft failure in situations of HLA-Bw4 incompatibility. These findings may be of future benefit in devising new antigen matching strategies for reducing the risk of humoral HLA allosensitization and chronic allograft rejection.

Matching for private or public HLA epitopes reduces acute rejection episodes and improves two-year renal allograft function.

The current role of HLA matching in renal transplantation is controversial. Public HLA epitope matching has been suggested to be as advantageous as private HLA matching, with the added benefit of increasing recipients' access to well-matched grafts. In this single-center study of 105 renal transplant recipients, we examined the association of HLA matching with early (0-3 months) and late (4-6 months) rejection episodes (RE), as well as renal allograft function up to 2 years after transplant. Poor HLA-DR, but not HLA-A or -B, matching was associated with early RE (0 DR matches, RE=2.7+/-0.19, 1 DR match, RE=2.37+/-0.18, vs. 2 DR matches, RE=1.5+/-0.38; P < 0.01). In contrast, poor HLA-B, but not HLA-A or -DR, matching was associated with late rejections (0 HLA-B matches, RE=1.1+/-0.51 vs. 1-2 HLA-B matches, RE=0.51+/-0.1; P < 0.004). HLA-B matching was also associated with a significantly lower serum creatinine (SCr) level at 24 months (0 HLA-B matches, SCr=178+/-20 micromol/L vs. SCr=132+/-6 micromol/L for 1-2 HLA-B matches; P < 0.025). Matching for 10 supertypic HLA-A and -B cross-reactive groups was associated with reduced late graft rejection (0-2 residue matches, RE=1.15+/-0.18 vs. RE=0.62+/-0.12 for 3 to 7 residue matches; P < 0.013) as well as a significantly lower SCr level at 24 months (0-2 residue matches, SCr=205+/-29 micromol/L vs SCr=131+/-6 micromol/L for 3 to 7 residue matches; P < 0.001) after transplantation. HLA-DR matching was associated with a reduced frequency of early rejection episodes, whereas HLA-B or residue/cross-reactive group matching was associated with a reduced frequency of late rejection episodes and improved graft function at 2 years.

Identification of antibodies toward private and public class I HLA epitopes in sensitized patients.

Antibodies to HLA determinants may decrease the increment after platelet transfusion and are correlated with increased rates of rejection in renal transplantation. Cross-match tests and HLA antibody specification are used to identify compatible donors for sensitized patients. However, search for cross-match negative donors may be ineffective, and many sera containing antibodies toward public HLA epitopes give no clear results in conventional specificity analysis. We tested a series of 4,625 sera from 1,073 patients with hematological, malignant or other diseases using a fluorescence lymphocytotoxicity test (LCT). We applied an evaluation program incorporating a list of public antigens belonging to known cross-reacting groups (CREGs) to detect antibodies toward private and just as well public epitopes. In 694 sera (15.0%) from 240 patients the panel reactivity (PRA) in LCT assay was higher than 5%. PRA was > or = 50% in 258 (37.2%) and < 50% in 436 sera (62.8%). In both groups we identified specific antibodies toward public HLA class I epitopes. Overall antibody specification was successful in 429 of 694 sera (61.8%) and in 175 of 240 patients (72.9%), respectively. The rate of antibodies against public epitopes shared by more than one HLA class I gene product was 203/694 (29.3%) with respect to tested sera and 83/240 (34.5%) with respect to patients. The rate of public epitope antibodies was highest in sera with PRA values from 30 to 90% showing public epitope specificity in 159 of 353 sera (45.0%). We conclude that antibodies toward public HLA class I determinants are detectable not only in highly sensitized patients. The described program may increase the rate of antibody specification and facilitate the platelet supply in platelet refractory patients.

Multiscreen serum analysis of highly sensitized renal dialysis patients for antibodies toward public and private class I HLA determinants. Implications for computer-predicted acceptable and unacceptable donor mismatches in kidney transplantation.

A multiscreen serum analysis program has been developed that permits a determination of antibody specificity for the vast majority of highly sensitized patients awaiting transplantation. This program is based on a 2 x 2 table analysis of correlations between serum reactivity with an HLA-typed cell panel and incorporates two modifications. One implements the concept of public HLA determinants based on the serologic crossreactivity among class I HLA antigens. The other modification derives from the premise that most highly sensitized patients maintain the same PRA and antibody profiles over many months and even years. Monthly screening results for patients with persistent PRA values can therefore be combined for analysis. For 132 of 150 highly sensitized patients with greater than 50% PRA, this multiscreen serum analysis program yielded information about antibody specificity toward public and private class I HLA determinants. The vast majority of patients (108 of 112) with PRA values between 50 and 89% showed antibody specificity generally toward one, two, or three public markers and/or the more common private HLA-A,B antigens. For 24 of 38 patients with greater than 90% PRA, it was possible to define one or few HLA-specific antibodies. The primary objective of the multiscreen program was to develop an algorithm about computer-predicted acceptable and unacceptable donor HLA-A,B antigens for patients with preformed antibodies. A retrospective analysis of kidney transplants into 89 highly sensitized patients has demonstrated that allografts with unacceptable HLA-A,B mismatches had significantly lower actuarial survival rates than those with acceptable mismatches (P = 0.01). This was shown for both groups of 32 primary transplants (44% vs. 67% after 1 year) and 60 retransplants (50% vs. 68%). Also, serum creatinine levels were significantly higher in patients with unacceptable class I mismatches (3.0 vs. 8.4 mg% [P = 0.007] after 2 weeks; 3.9 vs. 9.1 mg% [P = 0.014] after 4 weeks). Histopathologic analysis of allograft tissue specimens from 47 transplant recipients revealed a significantly higher incidence of humoral rejection (P = 0.02), but not cellular rejection, in the unacceptable mismatch group. These results suggest that the multiscreen program can establish which donor HLA-A,B mismatches must be avoided in kidney transplantation for most highly sensitized patients. For 18 of 150 high PRA renal dialysis patients, the multiscreen program could not define HLA-specific antibody. Most patients had greater than 90% PRA, and many of their sera appeared to contain IgM type nonspecific lymphocytotoxins that could be inactivated by dithioerythreitol (DTE).(ABSTRACT TRUNCATED AT 400 WORDS)

Recognition of an HLA public determinant (Bw4) by human allogeneic cytotoxic T lymphocytes.

The human class I HLA molecules are composed of both polymorphic, or private, and conserved, or public, regions. The private regions are recognized by alloreactive T cells and also serve as restriction elements for peptide presentation to autologous cells. Although the ability of public determinants to elicit antibody responses is well documented, little is known of their role in T cell function. In this study we examine the ability of one of these public HLA determinants, designated Bw4, to serve as a target Ag for CTL. We show that for some individuals the HLA-Bw4 epitope can function as a restriction element for CTL. This finding has important implications for organ transplantation.

Nucleotide sequence of the HLA-A26 class I gene: Identification of specific residues and molecular mapping of public HLA class I epitopes

A cosmid clone bearing an HLA class I gene has been isolated from a human genomic library by hybridization to a class I-specific probe. This clone encodes the HLA-A26 molecule characterized by immunologic reagents on murine transfected L cells. Nucleotide sequencing of the A26 allele has been performed, and the deduced amino acid sequence was compared with previously published HLA class I sequences. Amino acid sequence homologies between HLA-A26 molecules and members of the HLA-AW19 cross-reactive group were observed and allowed us to demonstrate that residue Q144 is the only critical residue involved in the binding of the 4E monoclonal antibody defining an epitope common to all HLA-B, -C, and -Aw19 alleles. This study also permitted designation of a V residue at position 189 in the third domain as possibly involved in the binding of the B1-23-2 monoclonal antibody. Furthermore, we located clusters of variability in reference to the three-dimensional structure of the HLA-A molecules, i.e., the ninth residue of the first β-strand domain, the upper surface of the first helical region, and both β and α structures of the α 2 domain.

Molecular mapping of a new public HLA class I epitope shared by all HLA-B and HLA-C antigens and defined by a monoclonal antibody

It has previously been shown that a mouse monoclonal antibody, designated 4E, reacts with an epitope common to all HLA-B and -C antigens and those of the HLA-Aw19 cross-reactive group, namely, HLA-A29, -A30, -A31, -A32, -Aw33, and -Aw74. In order to pinpoint the amino acid residues which comprise the public specificity recognized by 4E, and HLA-A29 cDNA clone was isolated and its predicted amino acid sequence compared with those of other cloned HLA class I genes. The isolated HLA-A29 cDNA corresponded to the rarer of the two A29 variant alleles, A29.1. Two amino acid residues of HLA-A29.1, gln-144 and arg-151, were found in all 24 HLA-B and HLA-C alleles examined but were present in only one of 15 HLA-A alleles for which sequence data are available. Importantly, this exceptional allele was HLA-A32, another member of the HLA-Aw19 cross-reactive group. Gln-144 and arg-151 should be capable of jointly contributing to the binding site for 4E, as they are situated in successive alpha-helical subregions and are predicted to be juxtaposed in the three-dimensional HLA molecule. Four other residues in the first or second external domains of HLA-A29.1 (thr-9, leu-62, gln-63, and his-102) were unique among the HLA-A alleles, but none of these was found in corresponding positions of HLA-B of -C alleles and thus failed to correlate with presence or absence of the 4E determinant. These observations are consistent with the notion that gln-144 and arg-151 define a determinant common to HLA-B, HLA-C, and the HLA-Aw19 cross-reactive group and the binding site of the monoclonal antibody 4E.

Molecular mapping of a new public HLA class I epitope shared by all HLA-B and HLA-C antigens and defined by monoclonal antibody 4E

Molecular mapping of a new public HLA class I epitope shared by all HLA-B and HLA-C antigens and defined by monoclonal antibody 4E

Individual differences in the cytotoxic T-lymphocyte response in man to public HLA determinants.

The allospecific anti-HLA response of cytotoxic T lymphocytes (CTL) from 22 unrelated individuals and 7 monozygous twin pairs was examined. From each responder, CTL were generated in several responder-stimulator combinations, each mismatched for one HLA-A or -B antigen. The CTL were assayed in the cell-mediated lympholysis (CML) on panels of third-party target cells, comprising cells that express the stimulating antigen (specific target cells), cells that express an antigen cross-reactive with the stimulating antigen (CREG target cells), and cells that do not express either the stimulating or a cross-reactive antigen (nonsharing target cells). Individual variations in the allo-CTL response were observed. We identified individuals (responders) who showed a consistently narrow CTL response and those who showed a broad reaction pattern to various stimulator cells. The narrow response was restricted almost entirely to specific target cells; the broad response comprised lysis of specific, CREG, and nonsharing target cells. These differences were evidently not dependent on the HLA-A, -B, -C phenotype of the responder, because HLA-A, -B, (-C)-identical individuals responded differently to the same stimulator. The identical response of monozygous twins indicates that the allogeneic CTL response is genetically controlled. The CTL response is not regulated by the HLA-DR antigens of the responder, nor is it influenced by the DR mismatch between responder and stimulator. The observed differences were not dependent on sex or age and could not be explained by differences in the T-lymphocyte subsets (OKT3+, OKT4+, OKT8+) or by differences in proliferative reactivity to mitogens (phytohemagglutinin, concanavalin A, phorbol-myristate acetate, pokeweed mitogen, anti-T3 monoclonal antibodies). The IL-2 activity in the supernatants of mixed lymphocyte cultures of broad and narrow responder-stimulator combinations did not differ.

Prediction of crossmatch outcome in highly sensitized dialysis patients based on the identification of serum HLA antibodies.

High levels of allosensitization (greater than 50%), which often occur in dialysis patients awaiting renal transplant, make donor selection difficult. Such patients may be included in elaborate protocols in which they are crossmatched with all available ABO compatible donors, or crossmatching may be deferred until a very-well-matched donor becomes available. The former approach of random crossmatching is costly and inefficient, while the latter approach may overlook crossmatch-compatible donors. We believe that the identification of antibodies present in highly reactive sera and the use of this information in donor selection would increase the frequency of crossmatch-negative donors for these patients. In this study eleven sera, reactive with 70% to 100% of a random cell panel, were obtained from multiply transfused dialysis patients. Sera were analyzed by standard (CDC) and antiglobulin augmented (AHG-CDC) lymphocytotoxicity, and by differential absorption with HLA-typed platelets. All sera contained only one or two antibodies directed against the high frequency public HLA epitopes, accounting for 85% to 100% of each serum's total reactivity. These characterized sera were crossmatched with 114 random normal donors. The frequency of negative crossmatches was 20.5%. However, if the serum antibody data had been used to preselect donors for crossmatch--that is, to exclude donors that were likely to be positive--the negative crossmatch frequency would have increased to 86.4%. The use of the serum analysis data in donor selection would have reduced the total number of required crossmatches by 78%. Serum analysis correctly predicted the outcome of 95.6% of crossmatches performed with an average of 3% false positives and 1.3% false negatives. This approach to donor selection reduces unnecessary crossmatching and increases the likelihood of finding crossmatch-compatible donors for highly reactive patients.

Pure interferon gamma enhances class II HLA antigens on human monocyte cell lines.

Human recombinant interferon gamma (IFN-gamma) with a chemical purity of over 90% was shown to enhance membrane expression of HLA-DR antigens on cells from 3 human myelomonocytic lines (HL-60, U-937 and THP-1). Immunofluorescence techniques using a series of anti-HLA-DR monoclonal antibodies showed that the low, although variable, levels of DR-positive cells were clearly enhanced as soon as 24 h after incubation with IFN-gamma. Only IFN-gamma was able to exert this effect, since incubation with high concentrations of IFN-alpha or -beta did not induce any significant modification of the percentage of HLA-DR-positive cells. In contrast, doses of IFN-gamma as low as 2 units were effective, indicating a highly preferential, apparently selective effect of IFN-gamma for enhancement of HLA-DR expression. Private class II antigen expression was also enhanced by IFN-gamma on the U-937 cell line. Through its enhancing effect on class II public and private HLA antigens on the membrane of human monocytes, the IFN-gamma lymphokine may have a critical role in the modulation of antigen presentation by monocytes and on the regulation of HLA-DR-restricted cell cooperation.

Description of a monoclonal antibody defining an HLA allotypic determinant that includes specificities within the B5 cross-reacting group

The selection and characterization of a cloned murine monoclonal antibody (4D12) that defines an HLA allotypic determinant is described. Antibody 4D12 immunoprecipitates HLA heavy and light chains. From studies on 4D12 reactivity with a large panel of well-characterized target cells, antibody 4D12 defines a public HLA determinant that includes specificities within but not identical to the B5 cross-reactive group. Antibody 4D12 should be useful in the study of HLA-B5-related supratypic antigens.

A public HLA antigen associated with HLA-A9, Aw32, and Bw4

The HLA complex codes for three distinct 44000 dalton molecules associated with beta2 microglobulin--HLA-A, B and C--each with its own multiallelic series of private antigens. The HLA-B molecule is exceptional in that it also carries a diallelic system, Bw4 and Bw6. One of these, Bw4, is often associated with the A-locus specificity A9. This finding has usually been ascribed to linkage disequilibrium between A- and B-locus antigens. We have shown, however, that an epitope called LHe is actually shared by A-locus and B-locus molecules. This epitope is found on all HLA-B molecules bearing the Bw4 determinant and is also found on all HLA-A molecules carrying the A9 (Aw23 and Aw24) or Aw32 specificities. We consider this a "public" HLA antigen; the possible molecular basis for both subtypic and public antigens on a single glycoprotein is discussed.

A monoclonal antibody defining a lymphoma-associated antigen in man.

A monoclonal antibody (Ab 89) was developed against a lymphoma-associated antigen on the tumor cells of a patient (N.B.) with a B cell, poorly differentiated lymphocytic lymphoma (D-PDL). By indirect immunofluorescence, Ab 89 was shown to react only with N.B. lymphoma cells and was unreactive with normal N.B. lymphoid cells, normal fractionated peripheral blood cells, other normal lymphoid tissues, fetal tissues, and non-lymphoid malignant cells. In addition, Ab 89 was unreactive with conventional immunoglobulins, private and public HLA antigens, or Ia-like antigens. More importantly, Ab 89 was reactive with some B cell lymphoid malignancies. Of the 57 B cell lymphomas tested, it was found that Ab 89 reacted with approximately 10% of B cell D-PDL and B cell chronic lymphocytic leukemia (CLL). Of interest was the finding that N.B. serum contained a circulating antigen which could specifically block the reactivity of Ab 89. The data obtained suggests that Ab 89 defines a tumor-associated antigen shared by a unique subgroup of B cell lymphomas. The group of patients reactive with Ab 89 did not fall into any histopathologic classification system. These data support the notion that there is greater heterogeneity of B cell lymphomas than may have been previously recognized.