Human leukocyte antigen (HLA) typing by DNA sequencing.

DNA sequencing is a powerful technique for identifying allelic variation within the human leukocyte antigen (HLA) genes. Sequencing is usually focused on the most polymorphic exons of the class I (HLA-A, -B, -C) and class II (HLA-DR, -DQ, and -DP) genes. These exons encode the antigen recognition site, the region of the HLA molecule that binds peptides and interacts with the T cell receptor for antigen and natural killer cell immunoglobulin-like receptors (KIR). Sanger sequencing of amplified DNA from each HLA gene from a preparation containing one or two alleles yields a sequence that is used to identify the alleles by comparison with a reference database.

The HLA-DP2 protein binds the immunodominant epitope from myelin basic protein, MBP85-99, with high affinity.

Myelin basic protein (MBP) is a candidate autoantigen in multiple sclerosis (MS). The immunodominant epitope for T-cell responses is assigned to the amino acid sequence MBP84-102, which binds to human leukocyte antigen (HLA)-DR2a (DRB5*0101) and HLA-DR2b (DRB1*1501) of the HLA-DR2 haplotype carrying the strongest genetic association with MS. In contrast with HLA-DR and -DQ molecules, HLA-DP molecules are poorly characterized with respect to the binding of self-peptides. We show here that HLA-DP2 binds MBP85-99 with high affinity, and that the amino acid residues in position MBP91, MBP92 and MBP93 are influencing the binding, as shown by alanine scans. We further used a series of truncated peptides to identify the core of the binding. Moving the frame along the peptide from residues 87-97 to 89-99 progressively decreased the binding affinity for HLA-DP2, while moving further towards the C-terminal completely abrogated the binding of peptides to HLA-DP2. The data suggest that the docking of the MBP85-99 peptide into the HLA-DP2 groove is dependent on MBP88V and MBP89V and may use either of them as primary anchor for the p1 position. HLA-DP2 might thus present the MBP85-99 peptide in the same register as the HLA-DRB1*1501, where the MBP89V is preferred as the p1 anchor. Notably, full-length MBP was able to compete for peptide binding with an affinity similar to that seen for the high-affinity binding peptides, DRα170-83 and IIP53-65. In summary, the HLA-DP2 molecule binds the immunodominant epitope in MS, MBP85-99, possibly in more than one register.

Identification of a novel DPA1 allele, DPA1*010602, in an East African population.

We report here a novel DPA1 allele, DPA1*010602, which was identified from an East African population during sequence-based human leukocyte antigen DPA1 typing. Through cloning and sequencing of multiple clones we confirmed that the new allele is identical to DPA1*010301 at exon 2 with the exception of two nucleotide substitutions (ATG to CAG) at codon 31. The substitutions changed the amino acid at codon 31 from methionine to glutamine. The World Health Organization nomenclature committee named the new allele DPA1*010602.

Sequence analysis of the MHC class II DPB1 gene in chimpanzees (Pan troglodytes).

The diversity of the MHC class II region in non-human primates is a focus of biomedical research because this region plays a crucial role in the recognition of antigens in the immune system. In particular, the chimpanzee [Pan troglodytes (Patr)], which belongs to the superfamily Hominoidea, has been used as a human model for the study of diseases such as human hepatitis C virus (HCV), human hepatitis B virus (HBV) and human immunodeficiency virus (HIV) infections, to which only humans and chimpanzees are susceptible. In the present study, polymorphisms of the MHC-DPB1 gene (Patr-DPB1) in a chimpanzee colony in Japan were examined using a stepwise polymerase chain reaction (PCR) technique. In order to design a suitable primer pair which would amplify exon 2 of the Patr-DPB1 gene, a fragment of approximately 8 kb from exon 1 to exon 3 was amplified from chimpanzee genomic DNA. After designing a 500-bp primer pair at the 3' region of intron 1 and the 5' region of intron 2, analysis of DPB1 exon 2 alleles of each chimpanzee was carried out. Twenty-two chimpanzees were used in our study, and we identified seven alleles by sequence analysis on the Patr-DPB1 gene, including one new allele. The obtained nucleotide sequence patterns suggest that Patr-DPB1 alleles emerge by genetic variations such as the exchange of sequence motifs and the accumulation of point mutations.

Beryllium binding to HLA-DP molecule carrying the marker of susceptibility to berylliosis glutamate beta 69.

Berylliosis is a chronic granulomatous disorder caused by inhalation of Be dusts that is driven by the accumulation of Be-specific CD4+ Th1-cells at disease sites. Susceptibility to berylliosis has been associated with the supratypic variant of HLA-DP gene coding for glutamate at position beta69 (HLA-DPbetaGlu69). The aim of this study was to test the hypothesis that the HLA-DPbetaGlu69 residue plays a role in the interaction with Be. To this end, soluble HLA-DP2 molecule (carrying betaGlu69) and its mutated form carrying lysine at position beta69 (HLA-DP2Lys69) were produced in Drosophila melanogaster and then used in a Be binding assays. BeSO4 (1-1000 microM) was used to compete for the binding of the biotinilated invariant chain-derived peptide CLIP (50 microM). BeSO4 was capable of compete out biotin-CLIP binding from the HLA-DP2 (IC50%: 4.5 microM of BeSO4 at pH 5.0 and 5.5 microM of BeSO4 at pH 7.5), but not from the HLA-DP2Lys69 molecule (IC50%: 480 microM of BeSO4 at pH 5.0 and 220 microM of BeSO4 at pH 7.5). Moreover, the binding of NFLD.M60, a MoAb recognizing an epitope in the HLA-DP peptide binding region, to the HLA-DP2, but not to the HLA-DP2Lys69 soluble molecules was inhibited BeSO4. NFLD.M60 binding to HLA-DP2, but not to HLA-DP2Lys69 stably transfected murine cells was also inhibited by Be both at pH 5.0 and at pH 7.5. The data indicate a direct interaction of Be with the HLA-DPGlu69 molecule, in the absence of antigen processing.

Jejuna of patients with insulin-dependent diabetes mellitus (IDDM) show signs of immune activation.

The roles of enteric viruses and food antigens as possible triggers in human insulin-dependent diabetes mellitus and the evidence that mucosal-associated homing receptors are important in both human and experimental diabetes prompted us to undertake an immunohistochemical study of intestinal specimens from patients with IDDM. We studied jejunal morphology and immunohistochemistry in 26 patients with IDDM, 13 of whom had the HLA-DQB1*0201 gene and therefore a higher risk of coeliac disease. The findings were compared with those in specimens from age-matched controls. Villous structure and the density of the intraepithelial lymphocytes were normal in every biopsy specimen. The extent of positivity with anti-DR and -DP antibodies in the villous epithelium was significantly greater in the specimens from patients than in those from controls (P = 0.0002 in both comparisons). The crypts were also more positive: for DR P = 0.0001, and for DP P = 0.002. The densities of T cells, CD4+, CD8+, and T cell receptor alpha/beta+ and gamma/delta+ cells in the epithelium and lamina propria were similar in patients and controls, but the patients had significantly more alpha 4/beta 7 integrin+ cells in the lamina propria (P = 0.006). No difference was seen between HLA-DQB1*0201-positive and -negative patients. These findings reflect a stage of inflammation in the structurally normal intestines of patients with IDDM and suggest secretion of inflammatory Th1-type cytokines in the intestine.

HLA-DP2: self peptide sequences and binding properties.

Although self peptides bound to HLA-DQ and, especially, HLA-DR allotypes have been described in some detail, few ligands that bind to HLA-DP have been identified. Toward this aim, naturally processed peptides were isolated from immunoaffinity-purified HLA-DP2 molecules expressed in cultured B lymphocytes. The size distribution of the peptide repertoire is generally similar to those reported for self peptides bound to HLA-DR and HLA-DQ molecules. Twelve peptides representing individual sequences including two nested sets were sequenced by mass spectrometry and/or N-terminal Edman analysis. Source proteins included MHC molecules and other integral membrane proteins as well as secretory and serum proteins. No dominant amino acid markers suggestive of particular enzymatic processing events were detected. Peptide specificity and affinity were examined in binding assays using synthetic peptides and purified HLA-DP and HLA-DR molecules. Anchor residues were tentatively assigned using alanine-substituted analogues of two self peptides. Some structural features of HLA-DP2 that may relate to peptide binding are considered.

Selective association of a 22-38 kDa glycoprotein with MHC class II DP antigen on activated human lymphocytes at the plasma membrane.

Two-dimensional electrophoretic analysis (2D-PAGE) of cell surface human DP and DR class II antigens identified a glycoprotein, designated pX, that is associated at the cell surface with DP but not DR class II antigen in activated T, B and NK lymphocytes but not in resting B lymphocytes, Raji B lymphoma cells, activated thymic epithelial cells or activated monocytes. pX is a heavily glycosylated protein with an apparent molecular mass spanning between 38 kDa and 22 kDa, that is reduced, after deglycosylation with Endo-F, to 22 kDa. The pX structure appears nonpolymorphic and independent of DP polymorphism, as suggested by 2D-PAGE migrational pattern of 125I-labelled Endo-F deglycosylated DP immunoprecipitates from T cells blasts derived from four donors with different DP allotypes. The apparent absence of polymorphism of pX is further suggested by two-dimensional peptide mapping of a single spot derived from 2D-PAGE of 125I-labelled DP deglycosylated immunoprecipitates from two donors.

HLA y diabetes mellitus insulino-dependiente / HLA and insulin-dependent diabetes mellitus

The propensity of an individual to develop type I (insulin dependent) diabetes mellitus is directly related to specipic HLA clase II proteins, specially those from DR and DQ regions. Genetic susceptibility to insulin dependent diabetes arises from a preestablished conformation of alpha and ß chains of DQ and ß chain of DR. Since the classic demonstration by McDevitt and colleagues that DQ ß chain aspartate at position 57 was protective against the development of the disease, many populations have been surveyed to study the association between the incidence Type I diabetes and determined frequencies of DR and DQ haplotypes. The assocation between these markers and susceptibility to Type I diabetes is well established in caucasians at the present time. However, little information is available for Latin American populations, that share a mixture of european, african and native genes. Our group is studying genetic markers of three Latin American populations (Argentina, Perú and Chile) and their possible association to the different incidence of Type I diabetes mellitus in each country

Differential surface expression of class II isotypes on activated CD4 and CD8 cells correlates with levels of locus-specific mRNA.

Class II molecules are encoded by genes located within the HLA-D region in man. Three major subregions (DR, DQ, and DP) have been identified, each containing several genes encoding the alpha- and beta-chains of the class II heterodimers. In man B lymphocytes constitutively express class II molecules, whereas T lymphocytes express class II molecules only after Ag activation. We have analyzed isotype-specific expression of class II on CD4+ and CD8+ T cell lines and clones derived from one donor after stimulation with allogeneic cells, with the soluble Ag (purified protein derivative of tuberculin), and with mitogen activation by PHA. Class II mRNA transcripts were analyzed in parallel by Northern blot hybridization with DR, DQ, and DP beta-chain-specific cDNA probes. Nearly all activated T lymphocytes expressed DR molecules and variable numbers of DP+ cells were detected in each T cell line or clone, regardless of the mode of Ag activation. DQ molecules were only found on T cell lines or clones of the CD4 phenotype. In contrast two CD8+ cytotoxic T cell lines did not bind DQ-specific mAb although they did express DR and DP molecules. The presence of DR, DQ, and DP mRNA transcripts correlated with the differential patterns of surface expression, indicating that variations in class II cell surface expression among activated T cells may relate to differential gene regulation in these cells.

Molecular characterization by high resolution isoelectric focusing of the products encoded by the class II region loci of the MHC in humans. II. DP alpha and DP beta gene variants.

To characterize the molecular polymorphism of the DP alpha and DP beta gene products, the HLA-DP molecules expressed by more than 200 cell lines were individually immunoprecipitated by using the mAb B7/21 and their neuraminidase-treated DP alpha and DP beta chains analyzed in IEF gels. These cell lines, most of them from members of 32 families, allowed the definition, by segregation analysis, of the IEF patterns of the DP polypeptide chains encoded by 129 distinct haplotypes. Both DP alpha and DP beta chains display polymorphic IEF-banding patterns. Two DP alpha (A and B) and seven DP beta (A, B, C, D, E, F, and G) IEF variants were characterized. The DP alpha B variant was found in linkage disequilibrium with both DP beta B and DP beta D. Linkage disequilibrium was also encountered with alleles at the DR and DQ loci. Finally, the correlations between the IEF DP alpha and DP beta variants and the primed lymphocyte test-defined HLA-DP specificities were determined by using a panel of 24 primed lymphocyte test-typed cell lines.

Human leukocyte antigen-DP in leukemia.

Human leukocyte antigen-DP (HLA-DP) typing was performed on patients with chronic myelogenous leukemia (CML, n = 44), acute nonlymphoblastic leukemia (ANLL, n = 34), or acute lymphoblastic leukemia (ALL, n = 41). Frequencies of DPw alleles in CML and ANLL patients were not significantly different from 254 controls, except that in ANNL DPw1 was absent. This was most likely due to the concurrent absence of DR3 with which DPw1 is in linkage. In contrast, in ALL, frequencies of DPw2 and DPw5 were significantly increased (corrected P less than 0.05, relative risk (RR) = 2.19 and corrected P less than 0.01, RR = 6.92, respectively). This was not due to linkage with DR. The frequency of DPw1 also tended to be reduced, but this was not caused by a similar decrease of DR3 in ALL. These results, therefore, demonstrate both positive and negative associations between major histocompatibility complex (MHC) gene products which are in only very weak linkage with the rest of HLA, and acute lymphocytic, but neither acute nor chronic myelogenous, leukemias. The HLA-DP region could thus contain long sought-after genes influencing susceptibility and resistance to leukemogenesis.

Detection of HLA-DP serological allodeterminants by the use of radioiodinated DP molecules.

HLA class II molecules were partially purified from cells of an HLA deletion mutant cell line, LCL721.82, that lost DR and DQ expression but retained DPw2 specificity and labeled with radioactive 125I. The radioiodinated preparation bound to DP-specific monoclonal antibody B7/21 as well as rabbit anti-HLA class II antiserum. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the component involved in these bindings gave, unlike known HLA class II molecules, a sharp and dense band of approximately 60 kDa under nonreducing conditions and a single but diffuse band of approximately 30 kDa under reducing conditions. By screening 401 anti-HLA class II alloantisera, including those distributed in the 9th International Histocompatibility Workshop and also those locally available, eight were found to possess significant binding activity. Specificity analysis of these eight binding-positive antisera on a panel of DP-pretyped HLA homozygous typing cells revealed the presence of two clusters, one corresponding to an allodeterminant associated with DPw1, 2 and 3 and the other to that associated with DPw2 and 4. These two determinants were shown by the sequential binding test to reside on the B7/21-defined HLA class II molecules. Thus, two major conclusions were drawn: two distinct allodeterminants are carried by a single DP molecule; and these serologically detected DP allodeterminants are supertypic to cellularly defined DP allospecificities.

Reactivity of human trophoblast with an antibody to the HLA class II antigen, HLA-DP.

Cytotrophoblast cells in term amniochorion and in first trimester chorionic villi were shown by immunohistology of frozen tissue sections to bind B7/21, an antibody specific for the MHC Class II antigen, HLA-DP. This binding was shown to be specific, as adsorption of the B7/21 antibody with a B cell line expressing HLA-DP prevented subsequent binding to trophoblast. When tested with a variety of other antibodies reacting with HLA-DR, HLA-DQ or the common sequences of HLA-DR, -DQ and -DP, trophoblast was negative, thus confirming previous reports. The significance of this unique pattern of reactivity of trophoblast is discussed.