HLA class I and H ferritin gene polymorphisms in normal subjects and patients with haemochromatosis

Summary The gene for idiopathic haemochromatosis is located on the short arm of chromosome 6 within 1 cM of the HLA-A locus. In this region there are many HLA class I genes, and there may also be a gene for the H subunit of ferritin. Both HLA class I and H ferritin genes are therefore candidates for the abnormal gene in idiopathic haemochromatosis. In 15 unrelated patients the frequency of HLA-A3 was 80% compared with 24% for 600 unrelated individuals from South Wales. The most common haplotype involved is probably HLA-A3, B7. DNA was prepared from leucocytes from 12 of these patients and from 85 normal subjects. After digestion with Taq1, electrophoresis, and Southern blotting, class I sequences were detected by hybridisation to an HLA class I probe (pHLA-A). Of the 34 restriction fragments detected, 22 were polymorphic. Particular fragments correlated with the presence of HLA-A antigens A1, 2, 3, 10, 11, w19, and 28, but there was little correlation with B antigens. Restriction fragment patterns specific for haemochromatosis were not found with TaqI or during less extensive studies with other restriction enzymes. No differences in restriction fragment patterns were found between four patients and four normal subjects apparently homozygous for HLA-A3 and B7. Examination of Southern blotting patterns for genomic DNA from patients and normal subjects with a panel of 12 restriction enzymes and a probe for the H ferritin gene (pDBR-2) revealed no polymorphisms associated with either idiopathic haemochromatosis or particular HLA phenotypes. These studies provide no support for either HLA class I genes or the H ferritin gene as candidates for the haemochromatosis gene.     

Molecular analysis of the human MHC class I region in hereditary haemochromatosis

Summary The unknown allele that predisposes to the development of haemochromatosis in man has been localized to the HLA class I region on the short arm of chromosome 6. We have utilized pulsed-field gel electrophoresis in conjunction with probes that map within, or in the vicinity of, this region to search for structural lesions that may further define the disease locus. Using the enzyme Mlu I, fragments that associated specifically with the HLA-A23, A31 and B8 alleles were identified. However, in members of three pedigrees affected by haemochromatosis, and in six unrelated patients with the disorder, no disease-specific differences were detected in the DNA fragments with four restriction enzymes and eight probes when compared with healthy individuals. These data suggest that the lesion responsible for hereditary haemochromatosis lies beyond the resolution of this technique and does not involve large structural deletions or extensive re-arrangements in this highly polymorphic region of the genome.     

Anonymous markers located on chromosome 6 in the HLA-A class I region: allelic distribution in genetic haemochromatosis

Summary Two yeast artificial chromosomes of the HLA class I region were subcloned. Four of the subclones studied displayed restriction polymorphisms that corresponded to six bi-allelic series. Allelic distribution of the anonymous markers was then studied by comparing a control population with a group of patients with familial haemochromatosis. Only one marker presents an unequivocal association with the haemochromatosis gene and is 100kb centromeric to HLA-A. This association however is not as strong as with HLA-A3. The results suggest two possible locations for the haemochromatosis gene: less than 100kb centromeric to the HLA-A locus, or on the telomeric side.     

HLA-linked rheumatoid arthritis.

Twenty-eight pedigrees were ascertained through pairs of first-degree relatives diagnosed with rheumatoid arthritis (RA). RA was confirmed in 77 pedigree members including probands; the absence of disease was verified in an additional 261 pedigree members. Pedigree members were serologically typed for HLA. We used likelihood analysis to statistically characterize the HLA-linked RA susceptibility locus. The genetic model assumed tight linkage to HLA. The analysis supported the existence of an HLA-linked RA susceptibility locus, estimated the susceptibility allele frequency as 2.16%, and estimated the lifetime penetrance as 41% in male homozygotes and as 48% in female homozygotes. Inheritance was recessive in males and was nearly recessive in females. In addition, the analysis attributed 78% of the variance within genotypes to genetic or environmental effects shared by siblings. The genetic model inferred in this analysis is consistent with previous association, linkage, and familial aggregation studies of RA. The inferred HLA-linked RA susceptibility locus accounts for approximately one-half of familial RA, although it accounts for only approximately one-fifth of the RA in the population. Although other genes may account for the remaining familial RA, a large portion of RA cases may occur sporadically.     

Intermediate inheritance of Tourette syndrome, assuming assortative mating.

Segregation analysis incorporating assortative mating was used to test for major locus inheritance of Tourette syndrome in a single large pedigree containing 182 members. The analysis provided evidence of a major locus with an intermediate inheritance pattern for which the penetrance was estimated from the data as 28% in heterozygotes and 98%-99% in homozygotes. A significant assortative mating correlation was estimated from the data as 70%-79%. In contrast, when assortative mating was not included in the model, intermediate inheritance was not inferred. If, in addition, constancy of the allele frequencies across generations was not assumed, Mendelian transmission was rejected. Each subject, affected or unaffected, was assigned a score reflecting the presence and severity of symptoms. Higher means scores in affected homozygotes than in affected heterozygotes suggested greater severity in homozygotes: genotype information was obtained from genotype probabilities computed assuming intermediate inheritance.     

HLA Class I Expression and Its Alteration by Preoperative Hyperthermo-Chemoradiotherapy in Patients with Rectal Cancer

Objective

Enhancing immunologic responses, including human leukocyte antigen (HLA) class I expression on tumor cells and recognition and elimination of tumor cells by tumor-specific cytotoxic T lymphocyte (CTL), is considered a novel concept of radiotherapy. The present study examined patients who underwent preoperative hyperthermo-chemoradiotherapy (HCRT) for locally advanced rectal cancer to assess the correlation between HLA class I expression and clinical outcome.

Materials and Methods

Seventy-eight patients with locally advanced rectal adenocarcinoma who received preoperative HCRT were enrolled. The median age of the patients was 64 years (range, 33–85 years) and 4, 18, and 56 patients had clinical stage I, II and III disease, respectively. Formalin-fixed and paraffin-embedded tissues excised before and after HCRT were subjected to immunohistochemical analysis with an anti-HLA class I-A, B, C antibody. HLA class I expression was graded according to tumor cell positivity.

Results

In pre-HCRT, the number of specimens categorized as Grade 0 and 1 were 19 (24%) and 58 (74%), respectively. Only 1 patient (1%) showed Grade 2 expression. However, 6 (8%), 27 (35%), 7 (9%), and 12 (15%) post-HCRT specimens were graded as Grade 0, 1, 2, and 3, respectively. There was a significant increase in HLA class I expression in post-HCRT specimens (p<0.01). However, neither pre- nor post-HCRT HLA class I expression affected overall survival and distant metastasis-free survival in clinical stage III patients. Univariate analysis revealed that Post-HCRT HLA class I expression showed a significant negative relationship with LC (p<0.05). Nevertheless, multivariate analysis showed that there was no correlation between HLA class I expression and clinical outcome.

Conclusion

HCRT increased HLA class I expression in rectal cancer patients. However, multivariate analysis failed to show any correlation between the level of HLA class I expression and prognosis.     

Mechanisms involved in NK resistance induced by interferon-gamma.

Human tumor cell lines were treated with interferon-gamma (IFN-gamma) and then used as target cells in NK assays to measure their ability to form conjugates and stimulate the production of NK cytotoxic factors (NKCF) and to determine their susceptibility to NKCF lysis. K562 and cell lines RS1, RS3, RS7, CAC, and CAP2, obtained from solid brain tumors, were used as targets, and peripheral blood lymphocytes (PBL) from normal donors were used as effector cells. IFN-gamma-treated cell lines had a decreased susceptibility to NKCF lysis and a decreased ability to induce the release of these factors without affecting target-effector cell binding. These results were not due to changes in HLA class I antigen expression, given that the level of HLA class I antigens on the tumor cell lines was not affected, the only exception being K562. In an attempt to further clarify the possible influence of HLA class I expression on K562, IFN-gamma-pretreated K562 cells were separated into HLA class I positive and HLA class I negative subsets for the NK assays. The results showed that both populations behaved similarly upon target-effector conjugate formation, whereas the HLA class I positive population showed a reduced susceptibility to lysis by NK cells and NKCF. Thus, these results establish that NK resistance induced by IFN-gamma is mediated by blocking the target cell's ability to activate NK cell triggering and release of NKCF and by blocking its susceptibility to lysis by these factors. This analysis helps to clarify not only the NK process but also the controversial regulatory effect of IFN in NK lysis.     

The HLA class I locus: analysis of RFLPs in hereditary hemochromatosis

The gene for hereditary hemochromatosis is linked to the HLA locus on chromosome 6. Four cloned DNA probes originating from the HLA class I region were used to detect seven restriction fragment length polymorphisms (RFLPs). Allele frequencies and segregation of each RFLP was determined. Analysis of RFLPs in 38 unrelated homozygotes with hemochromatosis revealed differences in allele frequencies between the control and the hemochromatotic groups but these differences did not reach statistical significance. Some differences persisted, however, even when only controls with the A3 antigen were compared with A3 hemochromatotics. Since both control and hemochromatotic groups were small, further studies will be necessary to ascertain whether these RFLPs could serve to locate the gene responsible for hereditary hemochromatosis.     

Clinical impact of HLA class I expression in rectal cancer

Purpose To determine the clinical impact of human leukocyte antigen (HLA) class I expression in irradiated and non-irradiated rectal carcinomas. Experimental design Tumor samples in tissue micro array format were collected from 1,135 patients. HLA class I expression was assessed after immunohistochemical staining with two antibodies (HCA2 and HC10). Results Tumors were split into two groups: (1) tumors with >50% of tumor cells expressing HLA class I (high) and (2) tumors with ≤50% of tumor cells expressing HLA class I (low). No difference in distribution or prognosis of HLA class I expression was found between irradiated and non-irradiated patients. Patients with low expression of HLA class I (15% of all patients) showed an independent significantly worse prognosis with regard to overall survival and disease-free survival. HLA class I expression had no effect on cancer-specific survival or recurrence-free survival. Conclusions Down-regulation of HLA class I in rectal cancer is associated with poor prognosis. In contrast to our results, previous reports on HLA class I expression in colorectal cancer described a large population of patients with HLA class I negative tumors, having a good prognosis. This difference might be explained by the fact that a large proportion of HLA negative colon tumors are microsatellite instable (MSI). MSI tumors are associated with a better prognosis than microsatellite stable (MSS). As rectal tumors are mainly MSS, our results suggest that it is both, oncogenic pathway and HLA class I expression, that dictates patient’s prognosis in colorectal cancer. Therefore, to prevent confounding in future prognostic analysis on the impact of HLA expression in colorectal tumors, separate analysis of MSI and MSS tumors should be performed. Frank M. Speetjens and Elza C. de Bruin contributed equally to this work. Cornelis J.H. van de Velde is the Chairperson of the Total Mesorectal Excision Trial.     

Cancerous HLA class I expression and regulatory T cell infiltration in gastric cancer

Background

Since antitumor immune reactions between tumors and intratumoral immunocytes have been verified in several human tumors, immunological therapeutic strategies must be considered to obtain the proper efficacy of tumor shrinkage under these conditions. Human leukocyte antigen (HLA) class I expression in cancer cells and degree of infiltration of regulatory T cells (Tregs) in the stroma have been regarded as important markers of antitumor immune reactions in the context of independent immunological mechanisms. In the current study, we investigated HLA class I expression and Treg cells infiltration in gastric cancer and discussed the clinical implications of this combinatory analysis in gastric cancer.

Patients and methods

A total of 141 gastric cancer patients who received R0 gastrectomy at Kagoshima University Hospital were studied. Immunohistochemically, in 141 gastric cancer patients, HLA class I expression and Treg cell infiltration in cancerous tissue were evaluated using HLA class I (EMR8-5) and forkhead box p3 (FOXP3) monoclonal antibodies. The correlation between clinical factors and tumor-infiltrating Treg cells was analyzed.

Results

HLA class I expression was positively associated with depth of tumor invasion (P?r?=?0.04). A better postoperative outcome was associated with fewer numbers of Treg infiltration (P?=?0.034). A combination of HLA and Treg analysis may lead to a more accurate prediction of postoperative outcome (P?=?0.02).

Conclusions

Two different antitumor immunological markers, Treg infiltration and HLA class I expression, affected clinicopathological factors in gastric cancer by different mechanisms. Thus, an immunological combination of HLA class I expression and Treg cell infiltration may more accurately predict postoperative outcome. Immunological balance needs to be restored after evaluation of each immunological deficit in gastric cancer.     

Increase in the expression of human leukocyte antigen class I in human fibroblasts by soluble factors secreted from human cytomegalovirus-infected cells.

The human cytomegalovirus (HCMV) is known to downregulate the expression of the human leukocyte antigen (HLA) class I for escape from immune surveillance. In order to understand the HCMV immune evasion mechanism, expression of HLA class I on the surface of HCMV-infected cells was investigated. A decrease in the HLA class I expression was observed at higher MOI; whereas at a lower MOI a slight increase in the HLA class I expression was observed. When HCMV-infected and uninfected cells were separately prepared on coverslips and co-cultured, the increased HLA class I expression was observed in uninfected cells. Treatment of the uninfected cells with the culture supernatant from HCMV-infected cells resulted in an increase in the HLA class I expression. A biochemical analysis of the HCMV-infected cell culture supernatant revealed the presence of interferon (IFN) beta interleukin (IL)-1beta, and IL-6. The HLA class I-enhancing activity of the culture supernatant was mimicked by IFN beta, but not by IL1-beta or IL-6, and was partially reversed by pretreatment with an antibody to IFN beta. Therefore, it appears that the HCMV infection of human foreskin fibroblast cells induces interferon beta and other soluble factor(s) that are responsible for the up-regulation of the HLA class I expression.     

Distinction between HLA class I-positive and -negative cervical tumor subpopulations by multiparameter DNA flow cytometry

BACKGROUND: The study of the molecular-genetic basis of heterogeneity of HLA class I expression in solid tumors is hampered by the lack of reliable rapid cell-by-cell isolation techniques. Hence, we studied the applicability of a flow cytometric approach (Corver et al.: Cytometry 2000;39;96-107). METHODS: Cells were isolated from five fresh cervical tumors and simultaneously stained for CD45 or vimentin (fluorescein isothiocyanate fluorescence), Keratin (R-phycoerythrin fluorescence), HLA class I (APC fluorescence), and DNA (propidium iodide fluorescence). A dual-laser flow cytometer was used for fluorescence analysis. Tissue sections from the corresponding tumors were stained for HLA class I antigens, keratin, vimentin, or CD45. RESULTS: Flow cytometry enabled the simultaneous measurement of normal stromal cells (vimentin positive), inflammatory cells (CD45 positive), epithelial cells (keratin positive), and DNA content readily. Normal stromal/inflammatory cells served as intrinsic HLA class I-positive as well as DNA-diploid references. Good DNA histogram quality was obtained (average coefficient of variation < 4%). Intratumor keratin positive subpopulations differing in HLA class I expression as well as DNA content could be clearly identified. Losses of allele-specific HLA class I expression found by immunohistochemistry were also detected by flow cytometry. CONCLUSIONS: We conclude that multiparameter DNA flow cytometry is a powerful tool to study loss of HLA class I expression in human cervical tumors. The method enables flow-sorting of discrete tumor and normal cell subpopulations for further molecular genetic analysis.     

Mutational analysis of the HLA class II interaction with Epstein-Barr virus glycoprotein 42

Entry of Epstein-Barr virus (EBV) into B lymphocytes requires the binding of viral glycoprotein 42 (gp42), a C-type lectin family member, to HLA class II. Recently, the structure of the gp42:HLA-DR1 complex was determined. In order to confirm the interaction as determined in the structural study and to identify other potential interactive residues, a mutational analysis of HLA class II was performed. A secreted form of gp42 (sgp42) reacted with a conformation-specific monoclonal antibody and blocked EBV infection. The binding of sgp42 and EBV entry to two sets of HLA class II mutants were tested. The first set of mutants were based on the known interaction of the C-type lectin Ly49A with HLA class I, and the second set of mutants were based on the identified interface in the gp42:HLA-DR1 complex. As expected, none of the mutants that would be predicted to interfere with the interaction of Ly49A with class I affected the interaction of gp42 with HLA class II, whereas mutants in amino acids identified in the gp42:HLA-DR1 structure inhibited sg42 binding to class II. In general, sgp42 binding correlated with efficient entry of EBV, as demonstrated by the necessity of glutamic acid 46 or arginine 72 in class II molecules. Furthermore, other HLA class II residues buried within the interface of gp42 and HLA class II when mutated had either no effect or a decrease in both binding and entry and implicate a region of class II important in stabilizing the interaction with gp42. These studies provide insight into the entry and fusion processes of the critical interaction between gp42 and HLA class II.     

Marked genetic polymorphism of the swine steroid 21-hydroxylase gene, and its location between the SLA class I and class II regions

Restriction fragment length polymorphism (RFLP) analysis of the swine 21-hydroxylase (CYP21) region was conducted on 31 unrelated SLA class I typed pigs, mainly Large Whites, including 15 haplotypes. Ten haplotypes were from SLA genotypic homozygotes and five were from SLA class I phenotypic homozygotes. DNA digestion with Hin dIII, TaqI and PstI, and hybridization to a 4.5-kb swine CYP21 genomic probe yielded respectively two, four and three RFLP patterns. Six patterns were identified with combined RFLP. In addition, analysis of the CYP21 region in families comprising several SLA recombinants demonstrated that the CYP21 gene lies in the DNA segment between the SLA class I and class II regions. These overall results reinforce our previous conclusion about the existence in the pig of a single 21-hydroxylase gene. The characterization of at least six CYP21 allelic patterns provides a new tool for studying the associations between the SLA region and zootechnical traits.     

Expression of class I HLA genes by trophoblast cells. Analysis by in situ hybridization

Evaluation of trophoblast cells by immunohistology has shown that subpopulations of trophoblast cells express class I HLA differently from one another and differently from embryonic and adult cells. Placental syncytial trophoblast does not express detectable levels of class I HLA; chorion membrane cytotrophoblasts bind one mAb to monomorphic determinants of class I Ag, W6/32, but not a second, 61D2. In the present study, sections of normal term placentae and matching extraplacental membranes were evaluated by in situ hybridization procedures for cells containing class I HLA mRNA using pHLA1.1, which is complementary to HLA-B. Class I Ag expression was identified by immunohistology using two mAb to class I HLA (W6/32, 61D2) and the mAb 4E to identify HLA-B. Placental syncytial trophoblast contained low to undetectable levels of class I mRNA and failed to bind all three mAb. Chorion membrane cytotrophoblast cells contained moderate levels of class I HLA mRNA and were positive with the mAb W6/32 but were negative with 61D2 and 4E. In adjacent tissues, fetal mesenchymal cells and maternal decidual cells contained high levels of class I mRNA and were positive with all three mAb. The results suggest that syncytial trophoblast may not express class I HLA because of low steady-state levels of class I HLA mRNA. In contrast, chorionic cytotrophoblast cells may express truncated versions of class I HLA or nonclassical HLA-A,B,C-like Ag. Regulation of the expression of class I HLA gene products may be essential to the development of a satisfactory immunologic relationship between the mother and her semiallogeneic fetus during pregnancy.     

Amnion membrane epithelial cells express class I HLA and contain class I HLA mRNA

Amnion epithelial cells in membranes from term deliveries, which have been reported not to express histocompatibility Ag, were evaluated for HLA by using an avidin-biotin immunoperoxidase staining system and for class I HLA mRNA by Northern blotting and in situ hybridization. There were three major findings from these studies. 1) Amnion cells frequently expressed class I HLA. Three mAb to monomorphic determinants of class I HLA were used: 61D2, PA2.6, and W6/32. 61D2 identified 1 of 8 fresh amnion membranes as class I positive whereas PA2.6 identified 4/8 and W6/32 identified 5/8. 2) Amnion cells contained class I HLA mRNA. RNA extracted from amnion membranes hybridized to a class I HLA probe (pHLA1.1) in Northern blotting. In situ hybridization procedures with pHLA1.1 showed that essentially all amnion cells contained class I HLA mRNA. 3) Levels of class I HLA mRNA in amnion cells could be modulated. Exposure of amnion explants to medium containing IFN-gamma enhanced levels of class I HLA mRNA in amnion cells, whereas epidermal growth factor diminished those levels. The results suggest that amnion cells transcribe class I HLA genes and are capable of synthesizing class I H chains but that expression may be modulated by extrinsic regulatory molecules.     

Ultra-high resolution HLA genotyping and allele discovery by highly multiplexed cDNA amplicon pyrosequencing

ABSTRACT: BACKGROUND: High-resolution HLA genotyping is a critical diagnostic and research assay. Current methods rarely achieve unambiguous high-resolution typing without making population-specific frequency inferences due to a lack of locus coverage and difficulty in exon-phase matching. Achieving high-resolution typing is also becoming more challenging with traditional methods as the database of known HLA alleles increases. RESULTS: We designed a cDNA amplicon-based pyrosequencing method to capture 94% of the HLA class I open-reading-frame with only two amplicons per sample, and an analogous method for class II HLA genes, with a primary focus on sequencing the DRB loci. We present a novel Galaxy server-based analysis workflow for determining genotype. During assay validation, we performed two GS Junior sequencing runs to determine the accuracy of the HLA class I amplicons and DRB amplicon at different levels of multiplexing. When 116 amplicons were multiplexed, we unambiguously resolved 99%of class I alleles to four- or six-digit resolution, as well as 100% unambiguous DRB calls. The second experiment, with 271 multiplexed amplicons, missed some alleles, but generated high-resolution, concordant typing for 93% of class I alleles, and 96% for DRB1 alleles. In a third, preliminary experiment we attempted to sequence novel amplicons for other class II loci with mixed success. CONCLUSIONS: The presented assay is higher-throughput and higher-resolution than existing HLA genotyping methods, and suitable for allele discovery or large cohort sampling. The validated class I and DRB primers successfully generated unambiguously high-resolution genotypes, while further work is needed to validate additional class II genotyping amplicons.     

Distribution of HLA class I altered phenotypes in colorectal carcinomas: high frequency of HLA haplotype loss associated with loss of heterozygosity in chromosome region 6p21

HLA class I loss or down-regulation is a widespread mechanism used by tumor cells to avoid tumor recognition by cytotoxic T lymphocytes, and thus favor tumor immune escape. Multiple mechanisms are responsible for these HLA class I alterations. In different epithelial tumors, loss of heterozygosity (LOH) at chromosome region 6p21.3, leading to HLA haplotype loss, occurs in 6–50% of all cases depending on the tumor entity. In this paper we report the frequency of LOH at 6p21 in 95 colorectal carcinomas (CRC) previously analyzed for altered HLA class I expression with immunohistological techniques. We used PCR microsatellite amplification of selected STR markers located on Chromosome 6 to identify LOH with DNA from microdissected tumor tissues and the surrounding stroma. Sequence-specific oligonucleotide analysis was performed in microdissected stroma and tumor cells for HLA typing, and to detect HLA haplotype loss. A high frequency (40%) of HLA haplotype loss was found in CRC. Eight tumors showed microsatellite instability. We sometimes observed two or more mechanisms responsible for HLA alteration within the same HLA-altered phenotype, such as LOH and HLA class I total loss. In 25 tumors (26%) no HLA class I alteration could be identified. These data are potentially relevant for CRC patients undergoing T-cell-based immunotherapy.     

Clustering HLA Class I Superfamilies Using Structural Interaction Patterns

Human leukocyte antigen (HLA) class I molecules are critical components of the cell-mediated immune system that bind and present intracellular antigenic peptides to CD8⁺ T cell receptors. To understand the interaction mechanism underlying human leukocyte antigen (HLA) class I specificity in detail, we studied the structural interaction characteristics of 16,393 nonameric peptides binding to 58 HLA-A and -B molecules. Our analysis showed for the first time that HLA-peptide intermolecular bonding patterns vary among different alleles and may be grouped in a superfamily dependent manner. Through the use of these HLA class I ‘fingerprints’, a high resolution HLA class I superfamily classification schema was developed. This classification is capable of separating HLA alleles into well resolved, non-overlapping clusters, which is consistent with known HLA superfamily definitions. Such structural interaction approach serves as an excellent alternative to the traditional methods of HLA superfamily definitions that use peptide binding motifs or receptor information, and will help identify appropriate antigens suitable for broad-based subunit vaccine design.