Peptide binding predictions for HLA DR,DP and DQ molecules

Background  

MHC class II binding predictions are widely used to identify epitope candidates in infectious agents, allergens, cancer and autoantigens. The vast majority of prediction algorithms for human MHC class II to date have targeted HLA molecules encoded in the DR locus. This reflects a significant gap in knowledge as HLA DP and DQ molecules are presumably equally important, and have only been studied less because they are more difficult to handle experimentally.     

Immunochemistry of the HLA class II molecules isolated from a mouse cell transfected with DQ alpha and beta genes from a DR4 haplotype

Cells from a mouse B lymphoma were transfected by DQ alpha and DQ beta genes derived from a DR4 haplotype. Quantitatively, the resulting expression of human class II molecules was similar to that of human B lymphoblastoid cell lines. Qualitatively, the transformant class II molecules differed from normal class II molecules in their carbohydrate moiety. As for their antigenic specificity, they were shown to carry two determinants previously identified on DQ molecules controlled by DR4 haplotypes, i. e., DQw3 and DCHON. The transformant molecules did not carry a third DR4-associated specificity, DC5 (equivalent to TA10), and must possess a structure allelic to DC5. However, no corresponding alloantigenic specificity was detected by a screening of relevant alloantisera.     

A strategy to determine HLA class II restriction broadly covering the DR, DP, and DQ allelic variants most commonly expressed in the general population

Classic ways to determine MHC restriction involve inhibition with locus-specific antibodies and antigen presentation assays with panels of cell lines matched or mismatched at the various loci of interest. However, these determinations are often complicated by T cell epitope degeneracy and promiscuity. We describe a selection of 46 HLA DR, DQ, and DP specificities that provide worldwide population (phenotypic) coverage of almost 90 % at each locus, and account for over 66 % of all genes at each locus. This panel afforded coverage of at least four HLA class II alleles in over 95 % of the individuals in four study populations of diverse ethnicity from the USA and South Africa. Next, a panel of single HLA class II-transfected cell lines, corresponding to these 46 allelic variants was assembled, consisting of lines previously developed and 15 novel lines generated for the present study. The novel lines were validated by assessing their HLA class II expression by FACS analysis, the in vitro peptide binding activity of HLA molecules purified from the cell lines, and their antigen presenting capacity to T cell lines of known restriction. We also show that these HLA class II-transfected cell lines can be used to rapidly and unambiguously determine HLA restriction of epitopes recognized by an individual donor in a single experiment. This panel of lines will enable high throughput determination of HLA restriction, enabling better characterization of HLA class II-restricted T cell responses and facilitating the development of HLA tetrameric staining reagents.     

Recombination between DQ alpha and DQ beta genes generates human histocompatibility leukocyte antigen class II haplotype diversity

Two major DR7 haplotypes have been defined on the basis of serologic typing: those that type as DQw2 and others that type as DQw3. In order to define the molecular basis for these serologic differences we have isolated and sequenced DQ alpha, DR beta I, and DQ beta cDNA clones from both representative haplotypes. These studies reveal that although the DQ alpha and DR beta I genes of both haplotypes are identical, the DQ beta genes are very different. These data suggest that the serologic differences of these two DR7 haplotypes are the result of a recombinational event that occurred between the DQ alpha and DQ beta genes. In addition, they emphasize the role of DQ recombination in generating "hybrid" HLA-DQ heterodimers.     

Complete characterization and sequence of an HLA class II DR beta chain cDNA from the DR5 haplotype

The human major histocompatibility complex includes the DP, DQ, and DR subregions, each of which contains at least one alpha chain gene and two beta chain genes. The products of the alpha chain gene and a beta chain gene from a given subregion combine to form a heterodimer which is found predominantly on the surface of immunocompetent cells, and is essential for effective cell-cell interactions and the generation of an immune response. The beta chain of the DR molecule is highly polymorphic, and it is this polymorphism which is thought to be ultimately responsible for the specific immune responsiveness and disease predisposition conferred by different DR molecules. While the sequences of DR beta chains of the homozygous DR1 cells, homozygous DR2, homozygous DR4, DR3/w6 cells and DR4/w6 genotypes have been partially or completely characterized, no sequence is yet available for the DR beta chain from a homozygous DR5 cell. A cDNA library was therefore constructed from the Swei cell line homozygous for the DR5 haplotype. A beta chain clone was isolated, characterized, and sequenced. Comparison with previously published DR beta chain restriction endonuclease maps and nucleotide sequences demonstrated that this clone was a DR beta chain clone. Comparison of the deduced amino acid sequence with other DR beta chain amino acid sequences shows three regions of variability in the first external domain, corresponding to amino acid residues 9-13, 26-38, and 67-74. The sequence of each of these variable regions in the beta chain from DR5 cells was identical or nearly identical to the sequences of variable regions found in the beta chains of other DR haplotypes, supporting the notion of gene conversion as an evolutionary mechanism generating polymorphism. The second external domain, and transmembrane and intracytoplasmic regions show a high degree of sequence conservation.     

Differential expression of HLA class II antigens in fetal human spleen: relationship of HLA-DP, DQ, and DR to immunoglobulin expression

Frozen sections of human fetal spleen from 12 to 20 wk gestation were examined by using polyclonal antibodies to Ig isotypes, monoclonal antibodies to HLA class II subregion locus products, B and T cells, and follicular dendritic cells. Scattered lymphoid cells in spleen sections from fetuses of 12 to 13 wk gestational age expressed IgM but not IgD. The appearance of lymphoid cells expressing IgD occurred at 14 to 15 wk before the formation of loose clusters of B cells at 16 wk. IgD expression was associated mainly with cells in these clusters, which by 17 wk had become definite follicles. Follicular dendritic cells were not detectable until 20 wk. OKT3-positive T cells were not detected until 17 wk, and at 20 wk constituted 5% of the nucleated cell population. HLA-DR- and DP-positive lymphocytes and macrophages were detectable in fetal spleen from 12 wk onward; DR was expressed on more cells than DP, and the numbers of cells stained by HLA-DR-specific monoclonal antibodies exceeded the number of Ig-positive cells in all spleens examined. HLA-DQ was expressed by consistently fewer cells than HLA-DR and -DP in all spleens tested. The small number of DQ-positive cells in spleens from 12- to 13-wk fetuses had the morphology of macrophages; HLA-DQ expression by lymphoid cells followed a similar pattern to IgD expression and was associated mainly with follicular lymphocytes. It could be demonstrated by double-labeling experiments that all follicular IgM-positive cells in 17- to 20-wk spleens expressed HLA-DP, DQ, and DR antigens: IgM-positive cells in 12- to 16-wk spleens and interfollicular IgM-positive cells in 17- to 20-wk spleens all expressed HLA-DR, but only 59% and 43% expressed DP and DQ, respectively. Ninety-one to 100% of IgD-positive cells in all spleens examined expressed HLA-DQ in addition to DR and DP. In these experiments IgD-negative, DQ-positive cells had the morphologic appearance characteristic of macrophages. These data suggest that class II antigens are differentially expressed on developing lymphoid cells; DR and DP expression occurring in the earliest spleens examined, with expression of DP on a subpopulation of DR-positive cells; IgD and DQ expression appears to be coincident on maturing B cells as they begin to form follicles. An immunoregulatory role for HLA-DQ in B cell development is implicated and remains to be fully investigated.     

Complete sequence of the HLA DQ alpha and DQ beta cDNA from a DR5/DQw3 cell line

The HLA-D region is composed of three subregions termed DR, DQ, and DP. We previously reported the sequence of a DR5 beta I and two DR5 beta III cDNA from the DR5 cell line Swei. We now report on the nucleotide and deduced amino acid sequence of the DQ alpha and DQ beta cDNA from the same DR5 cell line, which also types as DQw3. Comparison with other available DQ sequences indicates that DQ alpha has one region of major variability, whereas DQ beta appears to have four regions of variability. In addition, these comparisons indicate that DQw3 alpha from DR5 is different from DQw3 alpha from DR4, but identical to DQw2 alpha from DR3. In contrast, DQw3 beta from DR5 is very similar to DQw3 beta from DR4. These data indicate that at least for DQw2 and DQw3 it is the DQ beta chain that is responsible for DQ typing. Most sequence differences in DQ alleles can be attributed to point mutations; however, codon additions/deletions in the DQ alpha chain may contribute to variability. In addition, regions of possible gene conversion in the DQ alpha and DQ beta chains is suggested by the presence of a chi-like sequence in each chain. Finally, comparison of available haplotypes suggest recombination events may take place between DQ beta and DQ alpha, between DQ alpha and DR beta I, and between DR beta I and DR beta III.     

Transcriptional regulation of HLA class II and invariant chain genes

Class II (Ia) antigens are coded for by a family of genes located in the human MHC (HLA). These genes are regulated in a complex manner, being constitutively expressed, inducibly expressed, or not expressed, depending on the cell type examined. 6.1.6 is a variant of a normal B lymphoblastoid line that has lost expression of all class II molecules and has previously been shown to have a defect in the regulation of class II genes. In this report, we have examined those genes by Southern and Northern blotting and have found that 6.1.6 is severely deficient in mRNA for all class II genes examined, although the genes are structurally intact. P30, a partial revertant of 6.1.6, re-expresses mRNA for a subset of class II genes. mRNA for the class II-associated invariant chain is substantially reduced but not absent in 6.1.6.     

Complete nucleotide sequence of a functional HLA-DP beta gene and the region between the DP beta 1 and DP alpha 1 genes: comparison of the 5'' ends of HLA class II genes.

The complete nucleotide sequence of an HLA-DP beta 1 gene and part of the adjacent DP alpha 1 gene, up to and including the signal sequence exon, were determined. The sequence of the DP beta 1 gene identified it as the DPw4 allele. The six exons of the DP beta 1 gene spanned over 11,000 bp of sequence. The arrangement of the gene was broadly analogous to genes of other class II beta chains. The beta 1 exon was flanked by introns of over 4 kb. Comparisons with published sequences of cDNA clones indicated that an alternative splice junction, at the 3' end of the gene, is used in at least one allele. Variation in choice of splice junction indicates an additional mechanism for allelic variation in class II genes. The sequence also indicated that the DP beta 1 and DP alpha 1 genes are separated by only 2 kb at their 5' ends. Comparison of the 5' ends of the DP alpha 1 and beta 1 genes with other class II sequences, including the DZ alpha gene, showed conservation of several blocks of sequences thought to be involved in control of expression. Some areas of the introns were partially conserved in the DQ beta gene, and several other intron sequences were homologous to sequences found in other unrelated genes.     

Restriction fragment length polymorphism of DQ and DR class II genes of the bovine major histocompatibility complex

DQ alpha, DQ beta, DR alpha and DR beta class II genes of the bovine major histocompatibility complex (MHC) were investigated by Southern blot hybridizations using human probes. Hybridizations of these probes to genomic DNA, digested with PvuII or TaqI, revealed extensive restriction fragment length polymorphisms (RFLPs). The polymorphisms were interpreted genetically by analysing a family material, comprising five sires, 48 dams and 50 offspring, and a population sample comprising 197 breeding bulls. The analysis resolved 20 DQ alpha, 17 DQ beta, 5 DR alpha and 25 DR beta RFLP types. The segregation data were consistent with simple Mendelian inheritance of the RFLPs. The analysis of the bull sample showed that it is possible to apply the RFLP method for routine typing of class II polymorphism in population samples. The linkage disequilibrium in the DQ-DR region was found to be extremely strong as only about 20 DQ and about 30 DQ-DR haplotypes were observed despite the large number of possible haplotypes. Close linkage to the blood group locus M was also found; the M' allele occurred in strong linkage disequilibrium with the class II haplotype DQ1BDR alpha 4DR beta 1B. A population genetic analysis of the DQ data in the sample of breeding bulls revealed that the frequency of homozygotes was significantly lower than Hardy-Weinberg expectation and that the allele frequency distribution deviated significantly from the one expected for selectively neutral alleles.     

Discrimination between major histocompatibility complex class II DQ and DR locus products in cattle

We have used a panel of anti-major histocompatibility complex (MHC) class II monoclonal antibodies (mAbs) and have assessed their specificity for the products of the individual bovine MHC (BoLA) class II subregions. The mAbs identified two distinct class II molecules by affinity purification and ELISA. Two-dimensional immunoblotting confirmed these data and NH₂-terminal sequencing of the purified class II α chains of one member of each group identified the subregion specificity of the mAbs. The mAbs VPM36, TH22A and TH81A are specific for BoLA DQ, whereas VPM54, TH14B and J11 are specific for BoLA DR. SW73.2 reacts with both MHC subgroups of all cattle tested.     

HLA class II DR and DQ genotypes and haplotypes associated with rheumatic fever among a clinically homogeneous patient population of Latvian children

The HLA system is being paid more and more attention because it is very significant in polymorphous immunological reactions. Several studies have suggested that genetic susceptibility to rheumatic fever (RF) and rheumatic heart disease (RHD) is linked to HLA class II alleles. We hypothesized that HLA class II associations within RHD may be more consistent if analysed amongst patients with a relatively homogeneous clinical outcome. A total of 70 RF patients under the age of 18 years were surveyed and analysed in Latvia. HLA genotyping of DQA1, DQB1 and DRB1 was performed using PCR with amplification with sequence-specific primers. We also used results from a previous study of DQB1 and DRB1 genotyping. In the RF patients, HLA class II DQA1*0401 was found more frequently compared to DQA1*0102. In the RF homogeneous patient groups, DQA1*0402 has the highest odds ratio. This is also the case in the multivalvular lesion (MVL) group, together with DQA1*0501 and DQA1*0301. In the chorea minor patients, DQA1*0201 was often found. Significant HLA DQA1 protective genotypes were not detected, although DQA1 genotypes *0103/*0201 and *0301/*0501 were found significantly and frequently. In the distribution of HLA DRB1/DQA1 genotypes, *07/*0201 and *01/*0501 were frequently detected; these also occurred significantly often in the MVL group. The genotype *07/*0201 was frequently found in Sydenhamn's chorea patients that had also acquired RHD, but DRB1*04/DQA1*0401 was often apparent in RF patients without RHD. In the distribution of HLA DQA1/DQB1 genotypes, both in RF patients and in the homogeneous patient groups, the least frequent were *0102/*0602-8. The genotype DQA1*0501 with the DQB1 risk allele *0301 was often found in the MVL group. The genotype *0301/*0401-2 was frequently found in the RF and Sydenhamn's chorea patient groups. The haplotype *07-*0201-*0302 was frequently found in RF and homogeneous patient groups, including the MVL group. In addition, haplotypes *04-*0401-*0301 and *04-*0301-*0401-2 were frequent amongst patients with Sydenhamn's chorea. The protective alleles DQA1*0102 and DQB1*0602-8 in the haplotype DRB1*15 were less frequently found in RF patients. The results of the present study support our hypothesis and indicate that certain HLA class II haplotypes are associated with risk for or protection against RHD and that these associations are more evident in patients in clinically homogeneous groups.     

Class II genes of the human major histocompatibility complex. Comparisons of the DQ and DX alpha and beta genes

The human major histocompatibility complex, HLA, contains the genes of several class II molecules. We present here the molecular maps of the DQ and DX subregions and analyze the sequences of the polymorphic DQ alpha and DQ beta genes as well as the DX alpha and DX beta genes. The DQ alpha and DQ beta genes are oriented in opposite directions, approximately 12 kilobases apart. The DX alpha and DX beta genes are similarly oriented about 8 kilobases. The exon-intron organizations of the DQ alpha and DX alpha genes are analogous to those of other class II alpha genes. Comparison of the DQ alpha gene sequence to three DQ alpha cDNA clones shows that amino acid replacements are predominantly located between residues 45 and 80 in the amino-terminal domain. Analysis of the frequency of silent and replacement substitutions indicates that there is little selection against replacements in DQ alpha first domains. The exons encoding the second domains of DQ alpha and DX alpha are virtually identical, suggesting that a gene conversion event has occurred between these genes. The DX beta gene is very similar to the DQ beta gene but differs in the cytoplasmic portion. The DX beta gene contains a separate exon of 24 nucleotides encoding the core of the cytoplasmic tail. This exon is not expressed in the DQ beta genes due to a nonfunctional splice junction. Comparison of the number of nucleotide substitutions in the DQ beta first and second domain exons suggests that little or no phenotypic selection acts on the first domain whereas the second domain is under strong selection.     

HLA class II haplotype and sequence analysis support a role for DQ in narcolepsy

 A systematic haplotype and sequencing analysis of the HLA-DR and -DQ region in patients with narcolepsy was performed. Five new (CA)_n microsatellite markers were generated and positioned on the physical map across the HLA-DQB1-DQA1-DRB1 interval. Haplotypes for these new markers and the three HLA loci were established using somatic cell hybrids generated from patients. A four-marker haplotype surrounding the DQB1 ^* 0602 gene was found in all narcolepsy patients, and was identical to haplotypes observed on random chromosomes harboring the DQB1 ^* 0602 allele. Eighty-six kilobases of contiguous genomic sequence across the region did not reveal new genes, and analysis of this sequence for single nucleotide polymorphisms did not reveal sequence variation among DQB1 ^* 0602 chromosomes. These results are consistent with other studies, suggesting that the HLA-DQ genes themselves are among the predisposing factors in narcolepsy. Received: 18 March 1997 / Revised: 23 April 1997     

HLA-DR beta genes vary in number between different DR specificities, whereas the number of DQ beta genes is constant

Probes isolated from DR and DQ beta cDNA and genomic clones were used in hybridizations to restriction enzyme-digested DNA from human homozygous typing cells (HTC) as well as other DR homozygous cells in order to estimate the number of beta genes in the DR/DQ class II region. Varying numbers of DR beta genes were found in HTC of different DR specificities, from possibly one in DR 8 cells to three in cells of DR 2 to 7. The DR beta genes of different specificities seem to be related to one another in a distinct fashion. In contrast, all HTC contain two DQ beta genes per chromosome. The restriction site polymorphism of DQ beta genes is considerably more extensive than that of DQ serology, although one of the genes seems to be nonpolymorphic. In addition to the two DP beta genes identified previously, a minimum of three to five DQ and DR beta genes exist in the human haploid genome.     

Norrie disease gene is distinct from the monoamine oxidase genes

The genes for MAO-A and MAO-B appear to be very close to the Norrie disease gene, on the basis of loss and/or disruption of the MAO genes and activities in atypical Norrie disease patients deleted for the DXS7 locus; linkage among the MAO genes, the Norrie disease gene, and the DXS7 locus; and mapping of all these loci to the chromosomal region Xp11. The present study provides evidence that the MAO genes are not disrupted in "classic" Norrie disease patients. Genomic DNA from these "nondeletion" Norrie disease patients did not show rearrangements at the MAOA or DXS7 loci. Normal levels of MAO-A activities, as well as normal amounts and size of the MAO-A mRNA, were observed in cultured skin fibroblasts from these patients, and MAO-B activity in their platelets was normal. Catecholamine metabolites evaluated in plasma and urine were in the control range. Thus, although some atypical Norrie disease patients lack both MAO-A and MAO-B activities, MAO does not appear to be an etiologic factor in classic Norrie disease.