Quantifying Significance of MHC II Residues.

The major histocompatibility complex (MHC), a cell-surface protein mediating immune recognition, plays important roles in the immune response system of all higher vertebrates. MHC molecules are highly polymorphic and they are grouped into serotypes according to the specificity of the response. It is a common belief that a protein sequence determines its three dimensional structure and function. Hence, the protein sequence determines the serotype. Residues play different levels of importance. In this paper, we quantify the residue significance with the available serotype information. Knowing the significance of the residues will deepen our understanding of the MHC molecules and yield us a concise representation of the molecules. In this paper we propose a linear programming-based approach to find significant residue positions as well as quantifying their significance in MHC II DR molecules. Among all the residues in MHC II DR molecules, 18 positions are of particular significance, which is consistent with the literature on MHC binding sites, and succinct pseudo-sequences appear to be adequate to capture the whole sequence features. When the result is used for classification of MHC molecules with serotype assigned by WHO, a 98.4 percent prediction performance is achieved. The methods have been implemented in java (http://code.google.com/p/quassi/).

Functional classification of class II human leukocyte antigen (HLA) molecules reveals seven different supertypes and a surprising degree of repertoire sharing across supertypes.

Previous studies have attempted to define human leukocyte antigen (HLA) class II supertypes, analogous to the case for class I, on the basis of shared peptide-binding motifs or structure. In the present study, we determined the binding capacity of a large panel of non-redundant peptides for a set of 27 common HLA DR, DQ, and DP molecules. The measured binding data were then used to define class II supertypes on the basis of shared binding repertoires. Seven different supertypes (main DR, DR4, DRB3, main DQ, DQ7, main DP, and DP2) were defined. The molecules associated with the respective supertypes fell largely along lines defined by MHC locus and reflect, in broad terms, commonalities in reported peptide-binding motifs. Repertoire overlaps between molecules within the same class II supertype were found to be similar in magnitude to what has been observed for HLA class I supertypes. Surprisingly, however, the degree to which repertoires between molecules in the different class II supertypes also overlapped was found to be five to tenfold higher than repertoire overlaps noted between molecules in different class I supertypes. These results highlight a high degree of repertoire overlap amongst all HLA class II molecules, perhaps reflecting binding in multiple registers, and more pronounced dependence on backbone interactions rather than peptide anchor residues. This fundamental difference between HLA class I and class II would not have been predicted on the basis of analysis of either binding motifs or the sequence/predicted structures of the HLA molecules.

The presence of donor-specific human leukocyte antigen antibodies does not preclude successful withdrawal of tacrolimus in stable renal transplant recipients.

BACKGROUND: Because of the adverse events associated with the administration of immunosuppressive drugs, reduction of immunosuppression after solid-organ transplantation is highly desirable provided that graft rejection is prevented. In our transplant center, we used an immunosuppression reduction regimen in stable renal transplant patients. The presence of human leukocyte antigen (HLA) antibodies has been negatively associated with transplant outcome. Therefore, we evaluated the impact of HLA antibodies on the occurrence of acute rejection after immunosuppression reduction. METHODS: The presence and antigen specificity of HLA immunoglobulin G antibodies in serum samples were detected using enzyme-linked immunosorbent assay and single-antigen bead assays. Donor-specific cytotoxic potential was tested by standard CDC cross-match analysis. RESULTS: The presence of donor-specific or total HLA antibodies was not predictive for the occurrence of acute rejection after the reduction of immunosuppression. In addition, the presence of HLA antibodies did not preclude successful reduction of immunosuppression. After reduction of immunosuppression, newly formed HLA antibodies were seldom detected. Interestingly, evaluation of the cytotoxic potential of the detected HLA antibodies revealed that the one patient who developed donor-specific HLA antibodies and experienced a subsequent rejection episode was the only patient who carried cytotoxic HLA antibodies. This finding fueled the notion that the functional capacity, rather than the mere presence of donor-specific HLA antibodies, is indicative for transplant outcome. CONCLUSION: The presence of HLA antibodies does not preclude the successful reduction of immunosuppression in renal transplant patients with stable graft function.

Childhood asthma and human leukocyte antigen type.

Little is known about the relationship between human leukocyte antigen (HLA) class II genes and family history of asthma or atopy in relation to the incidence of childhood asthma. The objective of the study was to determine whether specific HLA class II genes (e.g., DRB1*03) are associated with asthma and whether such association explains the influences of family history of asthma or atopy on asthma incidence. A stratified random sample of 340 children who had HLA data available from the Rochester Family Measles Study cohort (n= 876) and a convenience sample of healthy children aged 5-12 years were the participants. We conducted comprehensive medical record reviews to determine asthma status of these children. The associations between the presence of specific HLA alleles and development of asthma and the role of family history of asthma or atopy in the association were evaluated by fitting Cox models. The cumulative incidence of asthma by 12 years of age among children who carry HLA DRB1*03 was 33%, compared to 24.2% among those who did not carry this allele. Adjusting for family history of asthma or atopy, gender, low birth weight, season of birth, HLA DRB1*04, and HLA DQB1*0302, the hazards ratio for HLA DRB1*03 carriers was 1.8 (95% confidence interval: 1.1-2.9, P= 0.020). We concluded that the HLA DRB1*03 allele is associated with asthma. However, the HLA class II gene does not explain the influences of family history of asthma or atopy on development of asthma. The mechanism underlying the association between asthma and HLA genes needs to be elucidated.

Associations between HLA class II alleles and type 1 diabetes mellitus in the Slovak population.

OBJECTIVES: Several associations between HLA complex and diabetes mellitus type IA were found in various groups of patients of Caucasoid population. This study was therefore prompted to be conducted in Slovak population, since any such has not yet been performed in Slovak population. METHODS: Patients suffering from DM-1A originated from all regions of Slovakia. Their age ranged from 1 to 42 years; but the criterion for including the subject to the study was the definition of diagnosis in older patients before their age of 15 (Table 1). The diagnosis was set up according to internationally accepted criteria. A total of 460 patients was typed for HLA-DQB1 alleles, among them 97 also for HLA-DQA1 and 146 for HLA-DRB1 alleles. HLA-typing was performed by a PCR-SSP method. Control group consisted of 196 (DQA), 143 (DQB1) and 130 (DRB1) unrelated blood donors aged 19-55 years old irrespective of their age or sex. The data obtained were expressed in a 2 x 2 contingency table and statistical significance was calculated by the Fisher exact test. RESULTS: Among 11 HLA-DQB1 alleles tested DOB1*0302 was the most frequent in DM-1A patients (30.33% vs. 5.59% in healthy subjects (HS), followed by DQB1*0201 (22.93% vs. 12.94%, respectively). In contrast, the frequency rate of DQB1*0301 (10.66% vs. 24.48%), DOB1*0602 (2.17% vs. 10.14%) and DQB1*0603 (2.5% vs. 8.39 %) were decreased in DM-1A patients. Out of 14 DQA1 alleles the highest occurrence rate showed DQA1*0301 (30.93% vs. 17.09) and DQA1*0501 (34.02% vs. 25.76%), while DQA1*0102 (8.76% vs. 16.58%) and DQA1*0201 (6.18 % vs. 13.51%7), respectively, were found to be the least frequent. Among 13 HLA-DRB1 alleles tested, the most common occurrence rates showed DRB1*03 (26.37% vs. 9.62%) and DRB1*04 (7.19% vs. 14.23%), while the least frequent alleles were DRB 1*15 (2.74% vs. 12.31%), DRB1*07 (7.19% vs. 14.23%), and DRB1*11 (2.74% vs. 20.38%). The alleles DQB1*0302 and DQA1*0301, respectively, were present in the same individual in all DRB1*04 positive patients, suggesting that they belong to the haplotype. Similar situation was observed with the alleles DQB1*0201, DQA1*0501, and DRB*0301, respectively, forming the second HLA haplotype so characteristic for DM1A.

In silico identification of supertypes for class II MHCs.

The development of epitope-based vaccines, which have wide population coverage, is greatly complicated by MHC polymorphism. The grouping of alleles into supertypes, on the basis of common structural and functional features, addresses this problem directly. In the present study we applied a combined bioinformatics approach, based on analysis of both protein sequence and structure, to identify similarities in the peptide binding sites of 2225 human class II MHC molecules, and thus define supertypes and supertype fingerprints. Two chemometric techniques were used: hierarchical clustering using three-dimensional Comparative Similarity Indices Analysis fields and nonhierarchical k-means clustering using sequence-based z-descriptors. An average consensus of 84% was achieved, i.e., 1872 of 2225 class II molecules were classified in the same supertype by both techniques. Twelve class II supertypes were defined: five DRs, three DQs, and four DPs. The HLA class II supertypes and their fingerprints given in parenthesis are DR1 (Trp(9beta)), DR3 (Glu(9beta), Gln(70beta), and Gln/Arg(74beta)), DR4 (Glu(9beta), Gln/Arg(70beta), and Glu/Ala(74beta)), DR5 (Glu(9beta), Asp(70beta)), and DR9 (Lys/Gln(9beta)); DQ1 (Ala/Gly(86beta)), DQ2 (Glu(86beta), Lys(71beta)), and DQ3 (Glu(86beta), Thr/Asp(71beta)); DPw1 (Asp(84beta) and Lys(69beta)), DPw2 (Gly/Val(84beta) and Glu(69beta)), DPw4 (Gly/Val(84beta) and Lys(69beta)), and DPw6 (Asp(84beta) and Glu(69beta)). Apart from the good agreement between known binding motifs and our classification, several new supertypes, and corresponding thematic binding motifs, were also defined.

Inflammatory glial activation in the brain of a patient with hereditary sensory neuropathy type 1 with deafness and dementia.

The brain of a patient with hereditary sensory neuropathy type 1 (HSN-1) associated with sensorineural deafness and early-onset dementia was neuropathologically investigated. Widespread neuronal degeneration in cerebral neocortex, hippocampus and basal ganglia was revealed, accounting for the clinical features. Loss of neurons with ballooning of residual neurons was remarkable in the hippocampus and frontal, parietal, and occipital lobes. Neuronal degeneration in these regions was accompanied by axonal dystrophy and glial reactions such as microgliosis and astrocytosis, however, only glial responses were prominent in the basal ganglia, brain-stem and cerebellum with mild neuronal loss. These results indicate that the widespread neuronal degeneration may be accelerated by inflammatory processes including glial activation in the brain of a patient with HSN-1 associated with deafness and dementia.

Immune responses to native beta(2)-glycoprotein I in patients with systemic lupus erythematosus and the antiphospholipid syndrome.

OBJECTIVE: To identify HLA class II associations with anti beta(2)-glycoprotein I (beta2GPI) antibodies in a cohort of Caucasian patients with systemic lupus erythematosus (SLE) and to determine whether these HLA genotypes act as restriction elements for lymphocyte proliferation to native human beta2GPI in vitro. METHODS: Anti-beta2GPI antibodies were detected in patient sera using enzyme-linked immunosorbent assays (ELISAs). HLA class II alleles (DRB1, DQB1) were determined by polymerase chain reaction-based DNA genotyping. In vitro peripheral blood mononuclear cell (PBMC) responses to native human beta2GPI were measured in a 7-day proliferation assay. RESULTS: We identified three groups of Caucasian SLE patients using these ELISAs. In group 1, 16 out of 18 SLE patients (89%) with anti-beta2GPI antibodies were positive for HLA-DRB1*0401/4/8, DR11 or DRB1*1302 (P=0.001 vs controls) compared with 23 out of 53 patients (43%) in group 2 with anti-cardiolipin antibodies only, 57 out of 151 patients (38%) in group 3 (SLE patients without anticardiolipin antibodies) and 109 out of 225 controls (48%). Fourteen patients with anti-beta2GPI antibodies had greater median stimulation indices to beta2GPI in vitro compared with the 15 controls studied (P=0.04). CONCLUSION: The HLA class II and PBMC proliferation data suggest that beta2GPI may be both a T- and B-cell autoantigen in SLE.

Constitutive expression of MHC class II genes in melanoma cell lines results from the transcription of class II transactivator abnormally initiated from its B cell-specific promoter.

In melanoma cell lines, two different patterns of MHC class II expression have been described, either an IFN gamma-inducible expression of HLA-DR and HLA-DP, with a faint or null expression of HLA-DQ, resembling that described for melanocytes, or a constitutive expression, i.e., IFN-gamma independent, of all three HLA-D isotypes. As this latter phenotype has been associated with a more rapid progression of melanoma tumors, we have analyzed in different melanoma cell lines the molecular mechanisms leading to this abnormal pattern of MHC class II expression. In agreement with the evidence of a coordinate transcription of the HLA-D genes in these cell lines, we have shown the constitutive expression of CIITA (class II transactivator) transcripts, CIITA being known as the master switch of MHC class II expression. Unexpectedly, these transcripts initiate from promoter III of the CIITA gene, a promoter that is mainly used constitutively in B lymphocytes. This expression was further shown to occur through factor(s) acting on the enhancer located upstream of CIITA promoter III, which was previously described in epithelioid cells as an IFN-gamma-response sequence. The hypothesis of a general abnormality of the IFN-gamma transduction pathway was dismissed. Constitutive transcription of CIITA from promoter III having been observed in unrelated melanoma cell lines, we propose the hypothesis that this phenomenon might not be a random event, but could be linked to the neoplasic state of the melanoma cells.

Isolation and mapping of the rabbit DM genes.

Proper peptide presentation by major histocompatibility complex (MHC)-encoded class II antigens is dependent on the products of the MHC DM loci. We identified the rabbit orthologues (RLA-DMA and -DMB) of human HLA-DMA and -DMB and found that they have 76.9% and 78.8% identity with HLA-DMA and -DMB, respectively. Like classical class II MHC genes, RLA-DM genes are more closely related to human HLA-DM genes than to mouse H2-DM. Among the DM family, there is a high degree of variability at the amino terminus of the DMa chains, and length variability in the cytoplasmic tails of both DMalpha and DMbeta. The rabbit DM genes are coexpressed with class II genes in lymphoid tissues, as are the DM genes of other mammals. The RLA-DM locus maps to the class II region of the rabbit MHC, and is flanked by the DP and DOB loci. Despite having some similarities to class II genes of bony fishes, the DM family represents a separate branch of the MHC class II family.

[Occurrence of D category VI in blood donors in Baden-Württemberg]. / Häufigkeit der D-Kategorie VI unter Blutspendern in Baden-Württemberg.

33,830 repeated donors and 9,157 first donors were screened for DVI. All D-positive first donors and the repeated donors with depressed D antigen (Du) were tested with Seraclone anti-D (IgM) that is known to be nonreactive with DIV. D-positive donors who were negative with Seraclone anti-D were further evaluated with a monoclonal IgG anti-D nonreactive with DIV. By using a D-screen panel it was attempted to type donors who were also negative with these antibodies. With this procedure, among 42,987 donors, 4 donors with DVI and 2 donors with probable DVI or closely related variants were detected. This is in agreement with previous reports from Great Britain and Australia. These donors with qualitative variants represent 2% of donors previously typed as Du and 10% of D-positive donors who were negative with Seraclone.

HLA-DR, DQ and DP typing using PCR amplification and immobilized probes.

A simple, rapid, and precise method of typing HLA class II polymorphism would be valuable in the areas of disease susceptibility, tissue transplantation, individual identification and anthropological genetics. Here we describe a method of analysing class II sequence polymorphism based on polymerase chain reaction (PCR) amplification and hybridization with oligonucleotide probes. One valuable property of sequence-based HLA typing strategies, like oligonucleotide probe hybridization, is that they reveal how and where two alleles differ, not simply that they can be operationally distinguished. The nature and location of HLA polymorphisms appears to be critical in disease association studies and are likely to be important in tissue typing for transplantation. New alleles at the DRB1, DPB1 and DQB1 loci are likely to be identified as this technology is applied to more and more samples, particularly in non-Caucasian ethnic groups. A new allele is uncovered as an unusual pattern of probe binding and then confirmed by sequencing. This pattern is observed because class II polymorphism is localized to specific regions and virtually all 'new' alleles have polymorphisms in the region of probe binding. Obviously, any new allele with a new polymorphic sequence in a region for which typing probes are not available would not be revealed by oligonucleotide typing. With the PCR primers and probes described here, 7 DQA1 alleles, 15 DQB1 alleles, 18 DPB1 alleles, and 32 DRB1 alleles are distinguished. Additional primers and/or probes can, of course, increase the allelic discrimination of oligonucleotide dot blot typing. These horseradish peroxidase (HRP)-labelled oligonucleotide probes are stable (greater than 2 years when stored at 4 degrees C) and the typing system is simple and robust. Over 500 samples from the CEPH pedigrees (unpublished data; A. B. Begovich, et al., manuscript in preparation) and greater than 1000 unrelated samples have been typed by this procedure. Although this dot blot/oligonucleotide hybridization procedure is a powerful and precise method of HLA class II typing, the complexity of the procedure increases as the number of probes required for analysis increases. The reverse dot blot method, based on an array of immobilized probes, allows the typing of individual samples in one single hybridization reaction. In this approach, a panel of unlabelled oligonucleotides are immobilized to a nylon membrane. The PCR product is labelled during the amplification reaction by using biotinylated primers and hybridized to the membrane. The presence of bound PCR product specifically hybridized to a given probe is detected using streptavidin-HRP conjugates and either chromogenic or chemiluminescent substrates.(ABSTRACT TRUNCATED AT 400 WORDS)

Optimizing PCR conditions for HLA class II SSO typing.

The polymerase chain reaction (PCR) has made the technique of sequence-specific oligonucleotide (SSO) typing fast, accurate and very sensitive. These combined techniques are an ideal tool for analysing the complex patterns of polymorphism seen throughout the HLA complex. The success of the technique relies heavily on accurate and specific amplification of the DNA under study. This paper considers the principles behind the PCR amplification technique and discusses the factors which lead to optimal amplification. Primer design is discussed and a variety of sources of target DNA considered. Precautions designed to prevent contamination are discussed. Reaction components are considered both in isolation and as part of the complete reaction. Finally, a complete PCR protocol is suggested. The paper is illustrated with examples of HLA class II amplification.