Properties of HLA class II molecules divergently associated with Goodpasture's disease

Goodpasture's disease provides an opportunity to analyse molecular mechanisms that may underlie MHC class II associations with autoimmune disease because it is caused by autoimmunity to a defined antigen [the 230 amino acid NC1 domain of the alpha3 chain of type IV collagen (alpha3(IV)NC1)] and has strong HLA class II associations. We compared the alpha3(IV)NC1 peptide binding of class II molecules with strong positive (DR15) and dominant negative (DR7/1) associations using an inhibition binding assay and short synthetic peptides spanning the sequence of alpha3(IV)NC1. DR15 in general bound the peptides with low affinity (three of 23 < 100 nM) compared to DR1 and DR7 (12 and 10 < 100 nM respectively), and no peptide bound DR15 with much higher affinity (>10-fold) than both DR1 and DR7. Thus DR15 molecules are unlikely to increase susceptibility to Goodpasture's disease by presenting a particular alpha3(IV)NC1-derived peptide uniquely well and DR1/7 are unlikely to protect by their inability to present particular peptides. However DR1/7 could protect by capturing alpha3(IV)NC1 peptides and preventing their display bound to DR15; the binding data suggest that all the major (biochemically detectable) alpha3(IV)NC1 peptides presented bound to DR15 by DR15 homozygous antigen-presenting cells (APC) would bind preferentially to DR1/7 in DR15, 1/7 heterozygote APC.     

Role of human papillomavirus in determining the HLA associated risk of cervical carcinogenesis.

AIMS--To investigate the role of human papillomavirus (HPV) in the association between HLA DQw3 and squamous cell cancer of the cervix (SCCC). METHODS--Tissue from 194 cervical samples, ranging from normal, through cervical intraepithelial neoplasia, to SCCC, were typed for HPV by amplification of the L1 gene using degenerate consensus primers, followed by oligonucleotide probing. HLA DQw3 typing was undertaken in the same samples using a new PCR amplification system using primers common to all DQ loci, followed by restriction digestion with Mlu 1 to differentiate HLA DQw3 types--null, heterozygous, and homozygous. The data were analysed using chi 2 analysis and by calculating relative risks with the 95% confidence interval. RESULTS--Samples (n = 188) were successfully typed for HPV and 177 were typed for HLA DQw3. There was a nonsignificant rise in the prevalence of HLA DQw3 in SCCC (64.3%) compared with the group with normal histology (53.2%). Analysis of the prevalence of HLA DQw3 on the basis of HPV infection rather than histology showed that 63 of 95 (66.3%) of the HPV positive samples contained HLA DQw3 alleles, compared with 39 of 78 (50.0%) of the HPV negative samples (chi 2 4.06; p < 0.05). CONCLUSIONS--There was a significant association between HLA DQw3 and cervical HPV infection. This may be because people with HLA DQw3 are less able to mount an effective immune response to HPV, which predisposes them to the development of SCCC.     

Soluble HLA-DR molecules in patients with HLA class II versus class I associated disorders

HLA genes have been identified as key genetic factors contributing to many chronic diseases characterized by autoimmune features. The role of HLA encoded molecules in the pathogenesis of these diseases is unresolved. We have now analysed soluble HLA-DR molecules circulating in the serum of patients with different autoimmune diseases and have defined parameters controlling serum levels. Patients with HLA-DR associated diseases were characterized by elevated serum concentrations of HLA-DR molecules and were clearly distinct from patients with HLA-B27 associated disorders. We did not find evidence for a correlation between disease activity, laboratory abnormalities and elevated serum concentrations of soluble HLA-DR molecules. Studies in normal individuals indicated that soluble HLA-DR molecules are at least partially regulated by the HLA haplotype. Highest serum concentrations were found in individuals carrying the HLA-DR3 or HLA-DR4 haplotype raising the possibility that the phenomenon of HLA-disease association reflects differences in the genetic control of soluble HLA-DR molecules. Interferon-gamma treatment caused an increase in serum concentrations of soluble HLA-DR molecules, whereas a decrease of circulating HLA-DR molecules was associated with an immunosuppressive with cyclosporine A. These data suggest that the patient's immunoresponsiveness represents a second important mechanism controlling circulating HLA-DR molecules.     

HPV16 E6 variants and HLA class II polymorphism among Chinese women with cervical cancer

Infection with human papillomavirus type 16 (HPV16) confers a high risk for the development of cervical cancer. Variants of this virus may interact differentially with host genetic factors, possibly affecting the disease pathogenesis. This study was designed to investigate the association between HPV16 E6 variants and human leukocyte antigen (HLA) polymorphism within a Chinese population. Peripheral blood from HPV16 positive Chinese women with cervical carcinoma, who had previously been tested for HPV16 E6 variants, was used for HLA class II typing. It was found that there was a significant positive association between DQB1*060101 allele and HPV16 As variant-positive cervical cancers (OR, 4.47; Pc=0.0018). A negative relationship was found between DRB1*150101-DQB1*0602 haplotype and decreased risk for HPV16 As variant-positive cervical cancers (OR=0.31; P=0.037). Similar tendency was observed for the haplotype DRB1*070101-DQB1*0201 with HPV16 As variant-positive cervical cancers (OR=0.16, P=0.024). Additionally, as for the HPV16 E6 prototype-positive cervical cancers, a significant positive association was found in DQB1*060101 allele (OR=5.95; P=0.002; Pc=0.036), and similar trends were observed for DQB1*030201 (OR=10.87, P<0.0001; Pc=0.0018), and DPB1*1301(OR=7.40, P=0.002; Pc=0.04). It was found that there was no significant association between DRB1-DQB1 haplotype and HPV16 prototype-positive cervical cancers. These data indicate that host genetic factors, such as HLA polymorphism, may determine the potential oncogenicity of the HPV16 E6 variant. The results suggest that a specific match between E6 variant proteins and HLA class II alleles may contribute to HPV16-related cervical carcinogenesis in a certain Chinese population.     

Frequent HLA class I loss is an early event in cervical carcinogenesis

Loss at chromosome 6p21.3, the human leukocyte antigen (HLA) region, is the main cause of HLA downregulation, occurring in the majority of invasive cervical carcinomas. To identify the stage of tumor development at which HLA class I aberrations occur, we selected 12 patients with cervical carcinoma and adjacent cervical intraepithelial neoplasia (CIN). We investigated HLA class I and beta2-microglobulin expression by immunohistochemistry in tumor and adjacent CIN. Loss of heterozygosity (LOH) was studied using microsatellite markers covering the HLA region. Fluorescent in situ hybridization with HLA class I probes was performed to investigate the mechanism of HLA loss. Immunohistochemistry showed absent or weak HLA class I expression in 11/12 cases. In 10 of these 11 cases, downregulation occurred in both tumor and CIN. Only in one case did the concomitant CIN lesion show normal expression. In 9/12 cases, LOH was present for at least one marker in both tumor and CIN, 1 case showed only LOH in the CIN lesion, and 1 case showed retention of heterozygosity for all markers in both tumor and CIN. We conclude that HLA class I aberrations occur early and frequently in cervical carcinogenesis. This might allow premalignant CIN lesions to escape immune surveillance and progress to invasive cancer.     

Characterization of monoclonal antibodies directed against HLA class II molecules

Four MAbs recognizing HLA class II antigens were produced by immunization with human leukemic cells, and were characterized through immunoprecipitation and cell distribution studies. They were tested against a panel of normal and leukemic cells, Epstein-Barr virus (EBV)-transformed homozygous typing cells (HTC)s, deletion mutant cell lines, and 11 other cell lines. The immunochemical studies revealed the presence of two bands of 34 and 28 kD, corresponding to the alpha- and beta-subunits of HLA class II antigens, respectively. The cell distribution studies led to the conclusion that GRB1, GRB2, and GRB3 MAbs recognize the HLA-DR monomorphic antigens, and that the GRB4 MAb seems to recognize basically the DR molecules but shows also a cross-reaction to DP molecules.     

Double-stranded deoxyribonucleic acid binds to HLA class II molecules and inhibits HLA class II-mediated antigen presentation

CD4⁺ T cells proliferating in response to purified double-stranded deoxyribonucleic acid (dsDNA) have been recently demonstrated in peripheral blood mononuclear cells of patients with systemic lupus erythematosus. Their activation was inhibited by anti-HLA class II (HLA-II) monoclonal antibodies; thus, the existence of a molecular interaction between dsDNA and HLA-II is conceivable. In this report we show that dsDNA specifically bind to HLA-II. After preincubating cells with purified dsDNA or synthetic oligonucleotides, dsDNA was detected on the cell membrane and in the lysates of HLA-II⁺ but not of isogenic HLA-II⁻ cell lines. We demonstrate that dsDNA binding inhibits that of a specific peptide to HLA-II. Mixed lymphocyte reaction and antigen-specific T cell proliferation were inhibited by the preincubation of stimulator cells or antigen-presenting cells with dsDNA. These results suggest the existence of a novel mechanism of down-modulation of the CD4⁺ T cell function generated by lack of stimulation due to the HLA-II presenting molecules being “n;-occupied” by dsDNA.     

HLA DPw1 class II molecules on human T cells

Human T cells express HLA class II antigens when activated by mitogens, alloantigens, or nominal antigens such as influenza virus. However, little is known about why they are expressed and the extent to which subsets of class II molecules (DR, DQ, and DP) are expressed. From studies with allocytotoxic antisera it is clear that DR and DQ molecules are expressed by T cells; cell surface expression of DP is more ambiguous because cellular typing methodologies are required. Alloreactive T-cell clones, specific for DPwl-associated antigens, were derived by limiting dilution in the presence of DPw1-positive stimulator PBL and IL-2 and screened on panels of DPw1-positive and DPw1-negative PBL's in proliferation assays. Clones that recognized DPw1-associated determinants were then assayed for responses using as stimulators, irradiated T-cell clones derived from a DPw1-positive donor. Of seven DPw1-specific clones, one (TLC 56.26) was highly responsive to the alloantigens expressed on the stimulator T-cell panel. Six such clones gave a range of lower responses to T cells although capable of recognizing DP-associated determinants on PBL stimulators.     

Affected sib-pair analysis of the contribution of HLA class I and class II loci to development of cervical cancer

Cervical cancer is a multifactorial disease and infection by oncogenic human papilloma viruses represents the main environmental risk factor. Only a subset of infections becomes persistent and develops into cancer, implying that genetic susceptibility factors are needed for malignant progression. Here, we use a population-based cohort of affected sib-pairs (ASPs) to examine the role of the human leukocyte antigen (HLA) class I and class II loci in cervical cancer susceptibility. Analysis of 278 ASPs revealed significant excess genetic sharing for all three HLA class II loci studied, DPB1, DQB1 and DRB1, with the strongest evidence for DQB1 and DRB1. No evidence of excess sharing was observed for the HLA class I HLA-B and HLA-A loci. When the material was stratified on the basis of the DQB1*0602/DRB1*1501 susceptibility haplotype, carriers showed significant sharing for all loci, whereas non-carriers showed no evidence of excess genetic sharing at any of the loci. However, for the DPB1 locus there was no difference in allele frequency between carriers and non-carriers indicating that the effect seen in DPB1 is not simply due to linkage disequilibrium. Our results show that the HLA class II represents a major genetic susceptibility locus to cervical cancer in contrary to the class I that do not appear to have a significant impact on predisposition to the disease. The strongest class II effects are coming from the DQB1 and DRB1 loci, but the DPB1 locus also contributes to the susceptibility to cervical cancer.     

HLA class II DNA analysis by RFLP reveals novel class II polymorphism

Polymorphism of the HLA-D/DR region has been defined by serologic and cellular methods. Additionally, protein and DNA analyses not only confirmed and refined the definition of the established polymorphisms but also revealed further polymorphisms for which no serologic or cellular correlate are known (yet). To study these in more detail, we analyzed the banding patterns obtained from Southern blot hybridizations with DRβ and DQ cDNA probes. Specific fragments reflecting already defined polymorphisms could be identified. A refined HLA-D/DR definition based upon the presence of DNA subtypes could be introduced. Fragments have also been identified that are associated with the DR/Dw specificities. Moreover, individuals with different DR types may also share fragments in hybridization assays. These shared hybridizing fragments (SHFs) are, for instance, found in individuals typed as DR4, DR5, DR7, and DRw8. In total, 20 SHFs were found using two restriction enzymes and the DRβ and DQ cDNA probes. Some of these SHFs correlate with antigenic determinants defined by broad reacting alloantisera, such as DRw52, but for 14 of these SHFs, no serologic equivalent has been found so far. Thus, SHFs reflect a conservation at the DNA level of the HLA class II region, which suggests that the polymorphic class II genes may be more conserved than previously thought. The possible biologic implications of conserved sequences in the HLA class II genes will be discussed.     

Polymorphism and evolution of HLA class I and II genes and molecules

Genes in the HLA complex, the human major histocompatibility complex (MHC), encode polymorphic HLA class I and II molecules that help T lymphocytes recognise and respond to foreign antigens. Certain HLA class I allotypes also regulate the response of natural killer cells. HLA class I and II molecules with little or no polymorphism contribute a variety of functions to the immune response, as do class I molecules coded by genes outside of the HLA region. Knowledge of the organisation of HLA class I and II genes, of the nucleotide sequences of their alleles, and the three-dimensional structures of their protein products, has facilitated analysis of the evolution and polymorphism of HLA class I and II genes and molecules. In turn, these analyses have provided insight into the mechanisms and selective forces driving change in the HLA complex.     

Distribution of HLA class II molecules in epidermal Langerhans cells in situ

We performed immunoelectron microscopic studies to investigate the expression of HLA class II molecules in Langerhans cells (LC). In the epidermis, LC expressed class II molecules on the plasma membrane of the dendrites, resulting in a class II positive reticulo-epithelial network, but not on the surface of the cell body. In contrast, isolated LC as well as activated LC in situ displayed an even and strong expression of class II molecules on their entire cell surface. Therefore, isolation and/or activation results in redistribution and up-regulation of class II molecules on the cell surface. In addition, double-labeling experiments were carried out with monoclonal antibodies to class II antigens and to LAMP-1, CD63 and α-glucosidase, specific markers for organelles of the endosomal/lysosomal system. The results show expression of class II molecules on intracellular, electron-dense vesicular structures, and co-localization of class II molecules and the markers for late endosomes and early lysosomes in human LC in situ. Expression of these markers was not found on Birbeck granules, an LC-specific organelle.     

Antibodies against denatured HLA class II molecules detected in luminex-single antigen assay

False-positive anti-HLA reactions may occur in Luminex-single antigen (SA) beads assays, and it is important to recognize them to correctly interpret the test. The purpose of this report is to describe a peculiar pattern of reactivity, characterized by positivity with beads coated with HLA-DRB1^∗09:01, DRB3^∗01:01, DRB3^∗02:02, DRB3^∗03:01, DPB1^∗02:01, DPB1^∗20:01 and DPB1^∗28:01, that was observed in 141 of 8121 serum samples tested in our laboratory with three different lots of the same kit (LABScreen® SA, One Lambda). These 141 serum samples came from 56 different patients on the kidney transplant waiting list, corresponding to 1% of the patients. Of these, 10 males had never been transfused or transplanted. About 66% of the patients had positive reactions against self-antigen HLA-DRB3 alleles. No reactions against native HLA-DRB1^∗09:01 were observed in flow cytometry crossmatch and in absorption/elution experiments, leading to the conclusion that the reactivity was due to antibodies against epitopes present in denatured forms of HLA-class II antigens. The occurrence of this reactivity pattern was associated with female gender and systemic lupus erythematosus (SLE).     

Molecular analysis of HLA class II polymorphism in Croatians

Abstract: HLA-class II polymorphisms have been studied in a population of 141 unrelated healthy Croatians using PCR amplification, followed by non-radioactive oligonucleotide hybridization. Thirty one DRB1, 8 DQA1, 13 DQB1 and 16 DPB1 alleles were found in the tested population. DRB1*1601, 0701, 1501, 0101 and 1104 are the most frequent alleles at the DRB1 locus. At the DQA1 locus two alleles predominate: DQA1*0501 and 0102, while the most frequent DQB1 allele is *0301. Analysis of HLA-DPB1 polymorphism showed that, as in other Europeans, DPB1* 0401 is the most frequent allele. Four different two locus haplotypic associations (DRB1-DRB3, DRB1-DRB5, DRB1-DQB1 and DQA1-DQB1) as well as three locus DRB1-DQA1-DQB1 haplotypic associations were assigned on the basis of known linkage disequilibria. Several unusual two-locus associations have been observed: DRB1*0301-DRB3* 0202, DRB1*1501-DRB5*02, DRB1*1601-DRB5*0101, DRB1*1502-DRB5*0101, DQA1*0103-DQB1*0503 and DQA1*0501-DQB1*0302. Among 236 examined DRB1-DQA1-DQB1 haplotypic combinations, the most frequent was DRB1*1601-DQA1*0102-DQB1*0502 that was found with statistically significant higher frequency than in other Europeans. Twenty-eight distinct probable haplotypes were observed just once, suggesting that the main characteristic of Croatian population is great heterogeneity of haplotypes. This study will serve as a reference for further anthropology studies, HLA and disease associations studies and for donor/recipient matching in organ and bone marrow transplantation.     

Are soluble monocyte-derived HLA class II molecules candidates for immunosuppressive activity?

Supernatants of human blood monocyte cultures suppressed PHA responses (IL-2 synthesis, IL-2R expression, DNA synthesis) of autologous and allogeneic lymphocytes. The main suppressive activity was found in the 65-kDa (and 23-kDa) range. It could be incompletely neutralized by mAb specific for a non-polymorphic HLA DR determinant and could also be adsorbed to and eluted from an anti-DR immunoabsorbent column. On blots of monocyte lysates and monocyte culture supernatants, the mAb RoDR recognized antigens of nearly the same Mr. The hypothesis that soluble HLA DR alpha beta heterodimers or beta chains are likely candidates for the suppressor factor was confirmed by analogous effects of purified HLA DR molecules. We favor a model in which soluble MHC class II molecules (in contrast to surface-bound ones) may interfere with the association and cross-linking processes necessary for T cell activation by competing for CD4 binding sites.     

HLA class II antigens are associated with Japanese pemphigus patients

We investigated the HLA class II antigens in 30 Japanese cases of pemphigus, 17 cases of pemphigus vulgaris (PV) and 13 cases of pemphigus foliaceus (PF), by both serologic and restriction fragment length polymorphism (RFLP) analyses. We detected two major haplotypes susceptible to PV, i.e., DRw12-DQw7 and DRw6-DQ5. In contrast, DR2 was absent in PV. RFLP analyses showed that DRw6 PV patients had a disease-associated restriction fragment representing DQw5, the same association as that found in DRw6 Jewish PV patients. However, DRw12 Japanese PV patients had DQw7, whereas DR4 Jewish PV patients had DQw8. On the other hand, all 13 PF patients were serologically typed for DQwl, which could not be further subdivided into DQw5 by RFLP analyses. These results suggest that Japanese and Jewish PV patients may be immunogenetically closely related to each other, but Japanese PV patients appear to be immunogenetically different from Japanese PF patients. (1991).     

Detection and quantitation of HLA class II molecules on keratinocytes by quantitative immunofluorescence

The paper describes measurements of HLA-DR antigen expression on normal human keratinocytes in culture using anti-HLA-DR antibodies and fluorescent Protein A or fluorescent second antibody methods and a low-light level video camera and image analysis program to quantitate the fluorescence output. The measurements are ultimately quantitated in terms of molecules of Ia antigen reacting material per cell. The method has a sensitivity at a S/N ratio of 10:1 of 180 molecules/micron2. The results show that normal keratinocytes do indeed express class II antigens on their surfaces at levels well above background. We confirm that treatment of the cells with gamma-interferon produces enhanced expression of DR antigens within 4 days. The method of quantitation is applicable to fluorescence and other low light level images.     

Identification of HLA-DRw52 associated antigens using HLA class II allogenotyping

The HLA-DR antigens DRw8, DRw11, DRw12, DRw13, DRw14 and DRw17 are strongly associated with the supertypic specificity DRw52. This association has been used to assist in assignment of serological specificity. However, difficulties in the identification of these antigens arise since they are serologically crossreactive. This report describes the application of restriction fragment length polymorphism (RFLP) allogenotyping to assist in the positive identification of DRw8, DRw11, DRw12, DRw13, DRw14, DRw17, DRw52a and DRw52b, and in addition describes further subtypes defined by RFLP which correlate with DQ or Dw associations. We also describe a novel TaqI/DR beta RFLP in a Caucasian family which types serologically as DRw12.