Ingrid Faé

Association between IgE response against Bet v I, the major allergen of birch pollen, and HLA-DRB alleles.

The association of the human IgE response against Bet v I, the major allergen of birch pollen, and the HLA-DR and DQ phenotype was studied. Birch pollen allergic patients showed a typical case history, positive skin-prick test, and positive RAST with birch pollen extracts. They were divided into two groups. Group I (n = 37) consisted of individuals generating IgE antibodies that selectively reacted with Bet v I. Their serum IgE did not react with minor allergens from birch pollen as tested by immunoblot analysis, nor did they show a response against allergens from a panel of grass and other tree pollen or perennial allergens from animals and fungi as determined by skin-prick test. Patients belonging to group II (n = 34) possessed IgE reacting with Bet v I plus one or more additional allergens. The control group consisted of 637 healthy blood donors. Comparison of the frequencies of RFLP-defined HLA-DR and DQ alleles in patients and the control group revealed that the distribution of DRB3 alleles in group I patients differed significantly from that in the control group: A higher frequency of the DRw52a/c alleles in comparison to the control group (pcorr less than 0.02) was observed. In addition, alleles defined by nucleotide sequences coding for the amino acid sequence tyrosine-phenylalanine-histidine at positions 30-32 of the beta chain of DR molecules were found with a higher frequency in patient group I (pcorr less than 0.02), too. These alleles comprise DRw52a/c and some DRB1 alleles.(ABSTRACT TRUNCATED AT 250 WORDS)

Impact of HLA class I high-resolution mismatches on chronic graft-versus-host disease and survival of patients given hematopoietic stem cell grafts from unrelated donors

Summary:There is consensus that matching of unrelated donors (URD) and patients for HLA class II alleles improves the outcome of hematopoietic stem cell transplantation (HSCT). However, the significance of HLA class I allelic mismatches for transplant outcome is under discussion and reports on long-term effects like chronic graft-versus-host disease (GVHD) are rare. Thus, we investigated the association of human leukocyte antigen (HLA) class I allele mismatches and outcome in 144 patients given HSCT from URD who were matched for HLA-DRB1, DRB3/4/5, and DQB1 alleles. The risk of chronic GVHD was significantly increased in patients with class I mismatched donors, the mismatch either detected by low- or high-resolution typing. A single HLA class I allele mismatch significantly increased the risk of chronic GVHD in multivariate analysis. Overall survival was significantly reduced in patient/donor pairs with more than one-allele class I mismatch. Thus, selection of unrelated donors for transplantation should be based on high-resolution HLA class I typing.

HLA typing by next-generation sequencing – getting closer to reality

Next generation sequencing (NGS) denotes novel sequencing technologies that enable the generation of a large number of clonal sequences in a single sequencing run. NGS was initially introduced for whole genome sequencing and for quantitation of viral variants or genetic mutations in tumor tissues; more recently, the potential for high resolution HLA typing and high throughput analyses has been explored. It became clear that the complexity of the HLA system implicates new challenges, especially for bioinformatics. From an economical point of view, NGS is becoming increasingly attractive for HLA typing laboratories currently relying on Sanger based sequencing. Realizing the full potential of NGS will require the development of specifically adapted typing strategies and software algorithms. In the present review, three laboratories that were among the first to perform HLA-typing using different NGS platforms, the Roche 454, the Illumina Miseq and the Ion Torrent system, respectively, give an overview of these applications and point out advantages and limitations.

KIR genes and KIR ligands affect occurrence of acute GVHD after unrelated, 12/12 HLA matched, hematopoietic stem cell transplantation

Interactions of polymorphic killer Ig-like receptor (KIR) receptors with KIR ligands have been shown to modify the outcome of hematopoietic SCT (HSCT). The association of these genetic factors with different transplantation endpoints, however, varies substantially, depending on clinical and study setup variables. We aimed to assess whether KIR ligands, KIR genes and KIR haplotypes are associated with HSCT outcome of 124 patients with various hematological malignancies, transplanted with 12/12 HLA matched grafts from unrelated donors. For this purpose, patient and donor KIR gene and KIR ligand polymorphisms were determined and correlated with clinical data in simple and multiple models. We found that a missing HLA-C2 ligand for donor inhibitory KIR2DL1 was significantly associated with an increased risk of acute GVHD (aGVHD) (II–IV) (hazard ratio (HR)=2.23, 95% confidence interval (95% CI): 1.21–4.10, P=0.010), as were the AA KIR haplotypes in patients and donors in HLA-C1CX (HR=2.37, 95% CI: 1.16–4.84, P=0.018) and in HLA-Bw4− (HR=3.20, 95% CI: 1.35–7.60, P=0.008) patients. On the contrary, transplantation of HLA-C1C2 patients with KIR2DS2 positive grafts were associated with a decreased risk of aGVHD (II–IV) (HR=0.24, 95% CI: 0.07–0.85, P=0.027). Thus, our single center study provides evidence for the modification of aGVHD risk by KIRs and their ligands.

Impact of HLA-DPB1 allelic and single amino acid mismatches on HSCT

The interpretation of the role of HLA‐DPB1 in unrelated haematopoietic stem cell transplantation (HSCT) is subject to discussion. We have investigated the role of HLA‐DPB1 allele matching in HSCT outcomes in 161 recipients who were HLA‐A, ‐B, ‐C, ‐DRB1 and ‐DQB1‐matched with their unrelated donors at the allelic level (10/10). In addition, we analysed the association of polymorphic amino acid mismatches of DPB1 molecule with HSCT end‐points, and a previously published permissiveness concept. HLA‐DPB1 allele mismatches were significantly associated with an increased incidence of acute graft‐versus‐host disease (aGvHD) and worse overall survival (OS). The mismatch at amino acid position 69 significantly increased the risk for transplant‐related mortality (TRM). Risk factors for aGvHD also included mismatches at positions 8, 9, 35, 76 and 84. This is to our knowledge, the first report of an in vivo effect of single amino acid mismatches on HSCT outcomes. In this study, grouping of allelic mismatches into permissive and non‐permissive categories and their association with transplantation end‐points was relevant for TRM but not for other clinical end‐points.

Association of HLA-E polymorphism with the outcome of hematopoietic stem-cell transplantation with unrelated donors.

HLA-E is a nonclassical human leukocyte antigen (HLA) class I gene with two antithetical products HLAE*0101 and HLA-E*0103. They are found on most tissues (1), HLA-E*0103 being expressed at considerably higher levels than HLA-E*0101. These differences depend on the affinity for available peptides and on the stability of refolded complexes (2). We correlated the HLA-E polymorphism with the outcome of hematopoietic stem-cell transplantation (HSCT) with unrelated donors. The median follow-up time of 124 patients transplanted at the University Hospital in Vienna between September 1995 and December 2005 was 80 months (range 35–158 months). Clinical endpoints were acute graft-versus-host disease (aGvHD) and chronic graft-versus-host disease (cGvHD), transplant-related mortality (TRM), relapse, and overall survival. Finally, 121 patients were evaluated for aGvHD and 102 for cGvHD. All patient donor pairs were matched for HLA-A, -B, -C, -DRB1, -DRB3/4/5, and -DQB1 alleles at four digit resolution. Patient and donor characteristics used in the statistical models are summarized in Table 1. Donor selection criteria, assessment of aGvHD and cGvHD, and supportive care during HSCT have been previously described (3). Statistical analyses comprised simple and multiple Cox regression models and competing risk analyses (4). HLA-E typing was performed by sequencing based typing of axon 3. The distribution of HLA-E alleles was similar in patients (HLA-E*0101: n 45, 37%; HLA-E*0101, *0103: n 53, 43%; HLA-E*0103: n 24, 20%) and donors (HLA-E*0101: n 38, 31%; HLA-E*0101, *0103: n 56, 46%; HLAE*0103: n 28, 23%). Two patients and two donors could not be typed due to missing DNA samples. Simple and competing risk analyses did not show evidence of an association of either of the analyzed genetic factors with clinical endpoints. Multiple regression analyses, however, showed associations with aGvHD (II–IV) (cumulative incidence at 80 days 41%, 95% confidence interval[CI]30–49), overall cGvHD (cumulative incidence at 3 years 39%, 95% CI 28–48), relapse (cumulative incidence at 3 years, 39% 95% CI 29– 48), and TRM (cumulative incidence at 180 days 17%, 95% CI 10–23). As to GvHD, HLA-E*0103, *0103 in donors was associated with a decreased risk of aGvHD (II–IV) (hazards ratio [HR] 0.39, 95% CI 0.16 – 0.99, P 0.047), whereas HLA-E*0101, *0103 in donors was associated with a decreased risk of overall cGvHD (HR 0.36, 95% CI 0.14 – 0.90, P 0.030). Regarding relapse, HLA-E*0103 alleles were associated with a higher risk (HR 2.24, 95% CI 1.03– 4.88, P 0.042). Concerning TRM, HLA-E*0103, *0103 in donors was a risk factor (HR 3.94, 95% CI 1.03– 15.02, P 0.045), whereas the presence of HLA-E*0101 alleles was protective (HR 0.32, 95% CI 0.11– 0.94, P 0.037). In summary, the presence of HLA-E*0103 in donors is associated with decreased risks of aGvHD (II–IV) and cGvHD and an increased risk of relapse. Regarding early posttransplant effects, HLA-E*0103 is associated with an increased risk of TRM. In addition to the genetic factors, multiple analyses showed significantly increasedriskofTRMinpatientswithaGvHD (I–IV), in patients at high-risk disease stage at transplantation, and in patients with donors older than 27 years (data not shown). Tamouza et al. (5) were the first to describe an association of HLA-E polymorphism with TRM in HSCT with unrelated donors. In their study, however, they found an association of HLA-E*0101, *0101 with increased TRM. This discrepancy might be due to differences between study populations (Tamouza et al. included also pediatric patients) and clinical protocols used. Especially, patients with bone marrow failure syndromes were more frequent in the Tamouza et al. study (27% vs. 0.8% in our patients). Patients suffering from bone marrow failure syndromes have high mortality rates after unrelated donor HSCT (6). The source of stem cells represents another difference (7). Although in our study, 56.5% of patients received peripheral blood stem cells, all patients in the study of Tamouza et al. had been transplanted with bone marrow-derived cells, known to prolong the time to hematologic and immunologic reconstitution and, therefore, prolong susceptibility to infections. Even though HLA-E*0103 allele cell surface expression exceeds that of HLAE*0101 (8), a similar correlation in the effectiveness of viral or bacterial antigen presentation has not been shown. Moreover, an impaired efficiency of HLA-E*0103 alleles in minor histocompatibility antigen presentation and T-lymphocyte stimulation hasbeenpostulated(9). Inefficientpresentation of minor histocompatibility antigens by HLA-E*0103 would be consistent with less aGvHD, an increased relapse rate, and increased TRM due to infections, as observed in our patients. Although our donor selection was based on a 12 of 12 allelic match, 47% of the pairs were mismatched for HLA-E alleles indicating different haplotypes. Therefore, it is possible that HLA-E serves as a surrogate marker for adjacent polymorphic loci that confer the effects we have observed. Katarina Ludajic Agathe Rosenmayr Ingrid Faé Gottfried F. Fischer Division of Blood Group Serology Medical University of Vienna Vienna, Austria

Specificities of Human CD4+ T Cell Responses to an Inactivated Flavivirus Vaccine and Infection: Correlation with Structure and Epitope Prediction

ABSTRACT Tick-borne encephalitis (TBE) virus is endemic in large parts of Europe and Central and Eastern Asia and causes more than 10,000 annual cases of neurological disease in humans. It is closely related to the mosquito-borne yellow fever, dengue, Japanese encephalitis, and West Nile viruses, and vaccination with an inactivated whole-virus vaccine can effectively prevent clinical disease. Neutralizing antibodies are directed to the viral envelope protein (E) and an accepted correlate of immunity. However, data on the specificities of CD4+ T cells that recognize epitopes in the viral structural proteins and thus can provide direct help to the B cells producing E-specific antibodies are lacking. We therefore conducted a study on the CD4+ T cell response against the virion proteins in vaccinated people in comparison to TBE patients. The data obtained with overlapping peptides in interleukin-2 (IL-2) enzyme-linked immunosorbent spot (ELISpot) assays were analyzed in relation to the three-dimensional structures of the capsid (C) and E proteins as well as to epitope predictions based on major histocompatibility complex (MHC) class II peptide affinities. In the C protein, peptides corresponding to two out of four alpha helices dominated the response in both vaccinees and patients, whereas in the E protein concordance of immunodominance was restricted to peptides of a single domain (domain III). Epitope predictions were much better for C than for E and were especially erroneous for the transmembrane regions. Our data provide evidence for a strong impact of protein structural features that influence peptide processing, contributing to the discrepancies observed between experimentally determined and computer-predicted CD4+ T cell epitopes. IMPORTANCE Tick-borne encephalitis virus is endemic in large parts of Europe and Asia and causes more than 10,000 annual cases of neurological disease in humans. It is closely related to yellow fever, dengue, Japanese encephalitis, and West Nile viruses, and vaccination with an inactivated vaccine can effectively prevent disease. Both vaccination and natural infection induce the formation of antibodies to a viral surface protein that neutralize the infectivity of the virus and mediate protection. B lymphocytes synthesizing these antibodies require help from other lymphocytes (helper T cells) which recognize small peptides derived from proteins contained in the viral particle. Which of these peptides dominate immune responses to vaccination and infection, however, was unknown. In our study we demonstrate which parts of the proteins contribute most strongly to the helper T cell response, highlight specific weaknesses of currently available approaches for their prediction, and demonstrate similarities and differences between vaccination and infection.

A Combination of Two Distinct in vitro Amplification Procedures for DNA Typing of HLA-DRB and -DQB 1 Alleles

The differential hybridisation of oligonucleotide probes to polymerase chain reaction (PCR)‐amplified DNA has become a standard procedure for tissue typing. We describe a typing method in which differential ligation replaces differential hybridisation, which is a significant simplification of this strategy. After amplification by the PCR two labelled, sequence‐specific oligonucleotides hybridise, in the fluid phase, to one strand of heat‐denatured amplification product in juxtaposition. In the case of perfectly complementary sequences surrounding the gap, a thermostable ligase catalyses the ligation of the two oligonucleotides, otherwise they stay separated. The use of heat‐resistant ligase enables easy repetition of the denaturation‐annealing‐ligation cycle in a thermocycler. The ligation products are detected by an enzyme linked immunosorbent assay. We tested this typing approach in a model system, the characterisation of three functional alleles of HLA‐DRB 3 using three probe pairs. No discrepancies were observed in typing 100 individuals of known genotypes. A total of 33 probe pairs combined with generic and group‐specific amplification allowed the typing of alleles of HLA‐DRB and ‐DQB1 loci at low resolution. We confirmed ligation‐based typing results of 259 individuals with sequence‐based HLA‐DRB1 typing and HLA‐DQB1 typing using PCR with sequence‐specific primers (SSPs). In addition, more than 1,500 ligation‐based HLA‐DRB1 typings were concordant with SSP typing. Excellent signal‐to‐noise ratios in the enzyme‐linked immunosorbent assay make ligation‐based typing remarkably robust. The time requirement of 2.5 h post‐PCR enables practicable typing of putative organ donors. The whole procedure is more easily amenable to automation than methods based on differential hybridisation requiring additional incubators and extra handling for hybridisation and washing.

Discrepant results of samples taken from different tissues of a single individual

Abstract Samples taken from different tissues of five bone marrow transplanted patients at least 5 years after successful bone marrow transplantation were investigated by STR-typing in order to find out whether or not donor cells can also be found in tissues other than blood. Donor alleles were detected in blood, buccal swabs and fingernails, but not in hair samples.

Correlation of sensitizing capacity and T-cell recognition within the Bet v 1 family

Background Bet v 1 is the main sensitizing allergen in birch pollen. Like many other major allergens, it contains an immunodominant T cell–activating region (Bet v 1142-156). Api g 1, the Bet v 1 homolog in celery, lacks the ability to sensitize and is devoid of major T-cell epitopes. Objective We analyzed the T-cell epitopes of Mal d 1, the nonsensitizing Bet v 1 homolog in apple, and assessed possible differences in uptake and antigen processing of Bet v 1, Api g 1, and Mal d 1. Methods For epitope mapping, Mal d 1–specific T-cell lines were stimulated with overlapping synthetic 12-mer peptides. The surface binding, internalization, and intracellular degradation of Bet v 1, Api g 1, and Mal d 1 by antigen-presenting cells were compared by using flow cytometry. All proteins were digested with endolysosomal extracts, and the resulting peptides were identified by means of mass spectrometry. The binding of Bet v 1142-156 and the homologous region in Mal d 1 by HLA class II molecules was analyzed in silico. Results Like Api g 1, Mal d 1 lacked dominant T-cell epitopes. The degree of surface binding and the kinetics of uptake and endolysosomal degradation of Bet v 1, Api g 1, and Mal d 1 were comparable. Endolysosomal degradation of Bet v 1 and Mal d 1 resulted in very similar fragments. The Bet v 1142-156 and Mal d 1141-155 regions showed no striking difference in their binding affinities to the most frequent HLA-DR alleles. Conclusion The sensitizing activity of different Bet v 1 homologs correlates with the presence of immunodominant T-cell epitopes. However, the presence of Bet v 1142-156 is not conferred by differential antigen processing.