A sensitive fluorometric assay for quantitatively measuring specific peptide binding to HLA class I and class II molecules

A sensitive, highly reproducible assay was developed for measuring binding of peptides to various HLA class I and II alleles. The assay is based on competition for binding to HLA between a peptide of interest and a fluorescent labelled standard peptide. This mixture is incubated with HLA to obtain equilibrium binding, and subsequently separated on an HPLC size-exclusion column in (i) a protein fraction containing HLA and bound peptide and (ii) a free peptide fraction. Each assay uses only 100 fmol labelled peptide and approximately 10 pmol of HLA. The analytical system contains an autosampler that samples from 96-well microtiter plates. Injections and data recording/evaluation is fully automated. Typical analysis time is 10–12 min per sample. The fluorescence in the HLA-bound peptide and free peptide containing fractions is measured on-line. The ratios of fluorescence signal in protein and peptide fractions at various concentrations of the peptide of interest are determined. IC₅₀ values are calculated from the binding curve as obtained by curve fitting of the data. Here we show results for peptide binding to HLA-DR1 and -DR17 molecules purified from detergent solubilized cell lysates, and for recombinant HLA-A^* 0201 and HLA-A^* 0301 expressed in E. coli.

The assay reported is sensitive and reproducible. It is non-radioactive and is non-labor intensive due to the high degree of automation.     

HLA class I and II polymorphisms in Azores show different settlements in Oriental and Central islands

Human leucocyte antigen-A, -B, -Cw, -DRB1, -DQA1 and -DQB1 polymorphisms were examined in the Azorean population. The data were obtained at high-resolution level, using polymerase chain reaction (PCR) with sequence-specific primer, PCR-sequence-specific oligonucleotides and sequence-based typing. The most frequent allele in each locus was: A*0201 (24.5%), B*510101 (9.8%), Cw*0401 (14.8%), DRB1*070101 (18.3%), DQA1*0201 (17.4%) and DQB1*0301 (19.4%). The predominant extended haplotype was A*0202-B*1503-Cw*0202-DRB1*090102-DQA1*0303- DQB1*0202 (1.9%), which was found to be absent in the Portuguese mainland. The present study corroborates historical sources that say the Azores were populated not only by Portuguese but also by other Europeans, mostly Flemish people. Despite dendrogram analysis showing some remote Asian genetic affinities, the lack of specific alleles and haplotypes from those populations does not allow us to conclude for direct influence. Haplotype and allele frequencies in Azores show no homogeneous distribution between Oriental and Central islands of this archipelago. The Oriental islands harbour several haplotypes already found in mainland Portugal and identified as Mediterranean and European. The Central group of islands on the contrary clearly shows an influence of north Europeans (most probably derived from a well-documented Flemish settlement), with much less affinity to mainland Portugal.     

Serological and molecular analysis of HLA class I and II alleles in Thai patients with psoriasis vulgaris

Abstract: The HLA class I and class II alleles in 67 patients with type I psoriasis vulgaris, 23 patients with type II psoriasis vulgaris and 140 healthy individuals were analyzed. The frequencies of HLA-A2, -B46, -B57 and DQB1*0303 were significantly increased in type I psoriasis compared to the controls (Pc<0.05). Molecular analysis of HLA-A2 alleles showed an increase in HLA-A*0207 and a decrease in HLA-A*0203 in type I psoriasis. HLA-DQBl*0301 was significantly decreased in type I psoriasis compared to the normal controls (Pc<0.05). No association of any alleles with type II psoriasis was observed. This data demonstrated two susceptible haplo-types: HLA-A1-B57-DRB1*0701-DQA1*0201-DQB1*0303 (AH57.1) and HLA-A2-B46-DRB1*0901-DQA1*0301-DQB1*0303 (AH46.1) for type I psoriasis in the Thai population. Besides, the haplotype AH46.1 was also associated with type II psoriasis.     

Molecular typing for HLA class I using ARMS-PCR: further developments following the 12th International Histocompatibility Workshop

Molecular typing for HLA class I was introduced in the 12th International Histocompatibility Workshop. Following a pilot study using three methods, sequence specific oligotyping (SSO), reverse dot blot and amplification refractory mutation system (ARMS)-PCR, the ARMS-PCR method was selected for use. A great advantage of an ARMS-PCR method is that, unlike the other two methods, it can determine whether sequence motifs are in cis or in trans, as ARMS-PCR detects two cis located motifs per reaction using forward and reverse sequence specific primers. Resolution was designed to be low to medium level for HLA-A, -B and -C alleles. Two hundred and fifty class I kits and 83 HLA-A2 subtyping kits were distributed. The A2 subtyping kit used a two round nested PCR system to identify all of the A2 alleles known at the time. Typing results on control DNA samples distributed with both the kits showed a very satisfactory performance. Since the 12th Workshop, the kits have been developed with the addition of new primers and primer mixes to increase the resolution of the test.     

Sequence-based typing of HLA class II DQB1

Due to the expanding number of known HLA class II DQB1 alleles, high-resolution oligotyping is becoming ineffective, therefore a sequence-based typing (SBT) strategy was developed to provide rapid and definitive typing of HLA-DQB1. HLA-DQB1*02, *03, *04, *05, and *06 alleles were individually amplified by polymerase chain reaction (PCR) using exon 2 group-specific primers. Forward and reverse PCR primers were tailed with M13 universal and M13 reverse sequences, respectively. Subsequent bi-directional cycle-sequencing was carried out using Cy5.5-labeled M13 universal primer and Cy5.0-labeled M13 reverse primer. Automated sequencing was performed in 30 min using a Visible Genetics, Inc. (VGI) MicroGene Clipper Sequencer. Full concordance was observed between this SBT method and oligotyping among 151 individuals.     

HLA class I DNA typing of 215 "HLA-A, -B, -DR zero mismatched" kidney transplants

DNA typing for HLA class II improves the typing quality and this was shown previously to be relevant for kidney graft survival. In this project we addressed the question whether molecular typing for HLA class I also increases the efficacy of HLA matching in kidney transplantation. 215 HLA-A,-B,-DR zero-mismatched donor/recipient pairs as defined by serological typing were selected. Retrospective HLA-A and HLA-B typing was performed both by the PCR-SSP and the PCR-SSOP method. DNA typing for HLA-A revealed discrepant results to serology in 5.7% of the donors and 2.8% of the recipients. HLA-B typing discrepancies were found in 6.6% of the donors and 5.6% of the recipients. 10.4% of the donors and 6.5% of the recipients showed either an HLA-A or an HLA-B discrepancy. Nearly one-third of the HLA-A discrepancies affected A19 splits. The most common reason for HLA-A discrepancies was the erroneous assignment of serological blanks, whereas HLA-B errors were caused mainly by the assignment of incorrect specificities. DNA typing allowed the definition of HLA-A and -B split specificities in all 118 "splitable" cases for which only broad specificities were reported based on serological typing. A total of 183 DNA class I compatible transplants had a 15% higher one-year graft survival rate than 32 transplants for which DNA typing revealed a class I incompatibility.     

Investigation of specimen mislabelling in paraffin-embedded tissue using a rapid, allele-specific, PCR-based HLA class II typing method

Mislabelling of surgical specimens can seriously affect the accuracy of histopathology reports. We describe two cases in which clinically suspected mislabelling was investigated by polymerase chain reaction (PCR)-based HLA DRB and DQB tissue typing of paraffin biopsy-derived DNA, using sequence specific primers (PCR-SSP HLA typing). In the first case, two patients underwent surgery for breast carcinoma. A subcutaneous lymph node containing metastatic carcinoma was received with the breast specimen from one patient, but was clinically considered more likely to originate from the other patient who underwent breast surgery on the same day. In the second case, histological examination of retained products of conception from a young woman revealed adenocarcinoma, but a repeat curettage specimen consisted of secretory phase endometrium. In case 1, PCR-SSP HLA typing confirmed the clinical suspicion that the subcutaneous lymph node received with tissue from one patient originated from the other patient. This result converted the first patient from lymph node positive breast carcinoma to lymph node negative disease. In case 2, there was no evidence from PCR-SSP HLA typing that the endometrial samples had originated from different patients. PCR-SSP HLA typing requires fewer steps than methods based on PCR amplification followed by oligonucleotide probing (PCR-SSOP HLA typing), and relies on the amplification of shorter sequences of DNA. Therefore, this technique can produce more rapid results than PCR-SSOP HLA typing, and is ideally suited to typing partially degraded DNA derived from formalin-fixed and paraffin-embedded tissue.     

Identification of woodchuck class I MHC antigens using monoclonal antibodies

Abstract: Two class I major histocompatibility complex (MHC) proteins with molecular masses of 43- and 39-kDa were identified in the cell surface membranes of normal woodchucks using a newly developed anti-woodchuck class I monoclonal antibody (mAb) B1b.B9 and immuno-blotting. B1b.B9 was generated by immunizing mice with viable wood-chuck peripheral blood mononuclear cells and was selected for anti-class I MHC reactivity using a cellular enzyme–linked immunoassay, indirect immunofluorescence on tissue sections and flow cytofluorimetry. The distribution pattern of class I MHC antigen on woodchuck lymphoid cells was found to be similar to that reported in other species. Also, the antigen expression on normal woodchuck hepatocytes was comparable to that observed on normal human liver parenchymal cells; thus, the antigen was not detected on hepatocytes by staining of liver tissue sections, but was found by indirect immunofluorescence staining of isolated liver cells. Western blot analysis of the plasma membranes from normal woodchuck hepatocytes revealed the presence of a single species of class I MHC heavy chain protein with a molecular mass of 43-kDa, whereas splenocyte plasma membranes showed intense expression of a 43-kDa species, as well as the presence of a 39-kDa protein. The 39- and 43-kDa proteins were extracted with Triton X-114 to the hydrophobic protein phase, suggesting that they both contain a hydrophobic transmembrane domain. The data obtained indicate that the B1b.B9 identifies a nonpolymorphic epitope of woodchuck class I MHC heavy chains, providing an important reagent for the study of the pathogenesis of hepatitis B virus infection in a woodchuck model.     

Possible impact of HLA class I and class II on malignancies driven by a single germ-line BRCA1 mutation

This study provides the first immunogenetic preliminary evidence that specific human leucocyte antigen (HLA) class I and class II alleles and haplotypes may be relevant for BRCA1 c.5263_5264insC driven oncogenesis. Observed HLA associations might have practical implications for establishment of predictive markers for the response to immunotherapies in malignancies driven by this germ-line mutation.     

HLA class I and class II DNA typing and the origin of Basques

Abstract: Seven HLA class I and class II loci (HLA-A, B, C, DRB1, DQA1, DPA1 and DPB1) were typed at the DNA level in two populations of the Iberian Peninsula (100 Basque and 88 Catalan individuals) in order to unravel their genetic relationship and to compare these results with other European and Mediterranean populations. For the first time,- the frequencies of alleles and haplotypes for the class I HLA loci at the DNA level in these populations are presented. The most frequent haplotype in both populations is A*29-Cw*1601-B*44-DRB1*0701-DQA1*0201-DPA1*0103-DPB1*0401. Neither population differed markedly from the highly homogeneous European and Mediterranean genetic landscape. The Basques, a European outlier population according to classical genetic markers, appear to lie within the genetic European variation with a slight uniqueness and show no clear relationship to North African populations, as has been postulated in some previous HLA studies. Here, the range of possibilities provided by the highly polymorphic HLA system is stressed by using genetic distances, phylogenetic trees and principal component analyses in order to reconstruct population history.     

Identification of a recurrent pattern of false-positivity by Luminex HLA MHC class I single antigen bead testing

Previously, a distinct MHC class II Luminex-single antigen bead (SAB) pattern was described and attributed to antibodies targeting denatured antigens. In this study, we describe a distinct MHC class I reactivity pattern observed in 1.8% (105/5992) of samples resulted in 2017. The pattern displays reactivity to the following Luminex-SABs: HLA-A*33:03, A*36:01, A*80:01, B*54:01, B*53:01, C*06:02, C*07:02, C*18:02, C*14:02, C*03:03, C*03:04, and C*15:02. This pattern was identified in patients with no sensitization history, negative FlowPRA results, and antibody to self-antigen(s). Epitope analysis failed to reveal a common determinant(s) to explain this pattern of reactivity. Additionally, we found this pattern to be prevalent in female patients (62%) and also those with systemic lupus erythematosus (62%). Given these findings, we speculate this pattern likely represents false-positive reactivity, possibly due to antibody targeting denatured antigens or a specific peptide, molecular mimicry, autoimmunity, or a combination thereof.     

HLA class II allele distribution in the Gypsy community of Andalusia, southern Spain

We have studied the allele distribution of DRB1, DQB1 and DPB1 loci in 80 unrelated Gypsies living in different eastern areas of the Andalusian province of Granada (southern Spain). The frequency distribution of HLA class II alleles and the genetic distance of Andalusian Gypsies from several Caucasian populations indicate a marked similarity - but not total - of the former with the Gypsy population previously studied in Madrid (central Spain), which suggests that both groups migrated together out of India. In terms of genetic distance, both Gypsy groups are more like the Czech Gypsies and the Northern Indian groups than their neighbouring Caucasian non-Gypsy populations. In summary our data support the hypothesis of a common anthropological origin of all three European Gypsy groups, which probably split up after their arrival in Europe.     

A novel HLA-A determinant recognized by a cytotoxic human hybridoma IgG1 monoclonal antibody (TrJ14)

Abstract: TrJ14 is a cytotoxic human IgG1Λ hybridoma mAb that recognized a novel HLA-A epitope expressed by lymphoblastoid B cells that are homo- or heterozygous for A2, A3, A11, A30, A31, A33, A68 and A69. Based on these results, the HLA type of cell line TEM (10w9057) was retyped as A66. When peripheral blood T cells isolated freshly from 265 HLA-typed normal individuals served as targets, TrJ14 killed cells expressing two TrJ14-positive HLA-A alleles, as well as the majority of cells having one TrJ14-positive and one TrJ14-negative HLA-A antigen. However, TrJ14 failed to recognize or reacted weakly with most HLA-A2 and -A3 heterozygous normal T cells when A2 or A3 was coexpressed together with a TrJ14-negative antigen. The serological reactivity of TrJ14 correlated with the amino acid valine and aspartic acid at positions 76 and 77 of the αl-domain helix. These amino acids were shared exclusively by all the identified TrJ14⁺ alleles.     

High-resolution HLA class I typing in the CEPH families: analysis of linkage disequilibrium among HLA loci

The HLA region on the short arm of chromosome 6 (6p21.3) contains the most polymorphic coding sequences in the human genome. High-resolution DNA-based HLA typing of population samples of the polymorphic class I loci, HLA-A, -B, and -C has only recently become feasible. Here, we report molecular HLA typing on family-based samples of European origin (the CEPH repository), which demonstrated very high polymorphism, with 20 A alleles, 38 B alleles and 19 C alleles in the sample of 248 independent haplotypes. In general, allele frequency distributions are consistently more even (lower observed homozygosity statistic) than expected from a past of selective neutrality suggesting a history of balancing selection. This was also true for the class II loci, DRB1, DQA1 and DQB1 in these samples, but not for the DPA1 and DPB1 loci, whose allelic frequency distributions were more skewed (higher observed homozygosity statistic) than expected under a neutral model. Although linkage disequilibrium is a prominent feature across the HLA region, only 19% of the eight locus haplotypes were sampled more than once. The relative age of some of the B alleles could be inferred from the pattern of B-C haplotypic associations. We suggest that the observed patterns of linkage disequilibrium reflect the operation of selection on nearly all HLA alleles.     

Prediction of spurious HLA class II typing results using probabilistic classification

While modern high-throughput sequence-based HLA genotyping methods generally provide highly accurate typing results, artefacts may nonetheless arise for numerous reasons, such as sample contamination, sequencing errors, read misalignments, or PCR amplification biases. To help detecting spurious typing results, we tested the performance of two probabilistic classifiers (binary logistic regression and random forest models) based on population-specific genotype frequencies. We trained the model using high-resolution typing results for HLA-DRB1, DQB1, and DPB1 from large samples of German, Polish and UK-based donors. The high predictive capacity of the best models replicated both in 10-fold cross-validation for each gene and in using independent evaluation data (AUC 0.820–0.893). While genotype frequencies alone provide enough predictive power to render the model generally useful for highlighting potentially spurious typing results, the inclusion of workflow-specific predictors substantially increases prediction specificity. Low initial DNA concentrations in combination with low-volume PCR reactions form a major source of stochastic error specific to the Fluidigm chip-based workflow at DKMS Life Science Lab. The addition of DNA concentrations as a predictor variable thus substantially increased AUC (0.947–0.959) over purely frequency-based models.     

Extended HLA haplotypes in Japanese homozygous typing cells

Abstract: We have defined extended HLA haplotypes including the HLA class II genes, the non-HLA genes such as TAP1, TAP2 and LMP2, and the (CTG)_n microsatellite repeats within the N0TCH4 gene between DRA and 21OH in 33 Japanese HLA homozygous typing cells (HTC). These conserved haplotypes characterized by unique linkage might be maintained as a result of functional co-operation among them in the antigen presentation pathway. These HTCs can be served as an original and ethnic-specific standard panel, providing useful genetic markers in haplotypic diversity, disease association, and anthropology studies.