HLA-F is a surface marker on activated lymphocytes.

Of the three nonclassical class I antigens expressed in humans, HLA-F has been least characterized with regard to expression or function. In this study, we examined HLA-F expression focusing on lymphoid cells, where our previous work with homologous cell lines had demonstrated surface HLA-F expression. HLA-F protein expression was observed by Western blot analysis in all resting lymphocytes, including B cells, T cells, NK cells, and monocytes, all of which lacked surface expression in the resting state. Upon activation, using a variety of methods to activate different lymphocyte subpopulations, all cell types that expressed HLA-F intracellularly showed an induction of surface HLA-F protein. An examination of peripheral blood from individuals genetically deficient for TAP and tapasin expression demonstrated the same activation expression profiles for HLA-F,but with altered kinetics post-activation. Further analysis of CD41+CD25+1 Treg showed that HLA-F was not upregulated on the major fraction of these cells when they were activated,whereas CD41+CD25- T cells showed strong expression of surface HLA-F when activated under identical conditions. These findings are discussed with regard to possible functions for HLA-F and its potential clinical use as a marker of an activated immune response.

Relationship between soluble MICA and the MICA A5.1 homozygous genotype in patients with oral squamous cell carcinoma.

NK and cytotoxic T cells play an important role in the elimination of virus-infected and tumor cells through NKG2D activating receptors, which can promote the lysis of target cells by binding to the major histocompatibility complex class I-related chain A (MICA) proteins. Polymorphisms in MICA may influence its binding to the NKG2D. The soluble form of MICA is released from the surface of tumor cells of epithelial origin. Whereas MICA expressed on the cell surface stimulates the immunoreceptor natural killer group 2, member D (NKG2D), the secreted form down-regulates NKG2D activity, thus allowing the tumor to escape immunosurveillance by NKG2D-expressing cells. In this study, we examined the association between MICA gene microsatellite polymorphisms and serum levels of soluble MICA in patients with oral squamous cell carcinoma (OSCC). We found that patients with OSCC were more likely to have the A5.1 allele when compared to healthy subjects and also more likely to be homozygous for this allele (p=0.041). Patients with the homozygous A5.1 genotype had higher levels of soluble MICA (p=0.031) and a lower survival rate (p=0.026).

Association between soluble MICA levels and disease stage IV oral squamous cell carcinoma in Japanese patients.

The soluble form of major histocompatibility complex class I-related chain A (MICA) is released from the surface of tumor cells of epithelial origin. Although MICA expressed on the cell surface stimulates the immunoreceptor natural killer (NK) group 2, member D (NKG2D), the secreted form downregulates NKG2D activity, thus allowing the tumor to escape immunosurveillance by NKG2D-expressing cells. In this study, we examined the association between serum levels of soluble MICA and the severity of disease in patients with oral squamous cell carcinoma (OSCC). We used enzyme-linked immunoabsorbent assay to measure serum levels of soluble MICA in OSCC patients and normal control individuals. Among patients categorized according to most disease parameters tested (tumor size, location, grade of differentiation, regional lymph node status, disease stage), soluble MICA levels in sera did not statistically differ from those in normal control individuals. Patients with stage IV disease and/or regional lymph node metastasis did, however, exhibit significantly higher serum levels of soluble MICA than control individuals (95% confidence interval (CI), 0.65-2.45, p = 0.021, and 95% CI, 0.62-4.42, p = 0.031, respectively). Overall survival rates were significantly higher for OSCC patients with low soluble MICA levels (<50 pg/ml) than for those with high soluble MICA levels (>50 pg/ml) (95% CI, 0.43-2.75, p = 0.03). Serum levels of soluble MICA may be useful in the diagnosis of advanced stage OSCC and as an indicator of regional lymph node metastasis.

Soluble HLA-G is absent from human embryo cultures: a reassessment of sHLA-G detection methods.

In recent years, the number of patients receiving in vitro fertilization (IVF) has been increasing, though the rate of successful implantations has remained at 10-20%. A major goal of this procedure is to afford the ability to select embryos with the most potential for implantation and development. Previous studies claimed to have detected soluble HLA-G (sHLA-G) protein in culture supernatant from 2 to 3-day embryos using ELISA methods, and concluded that sHLA-G protein levels were associated with successful implantation. This result, if substantiated could provide an important tool for IVF. In this study, we have re-examined these experiments by attempting to detect sHLA-G in the medium from 2 to 3-day embryos (84 samples) and 4 to 6-day embryos (25 samples) in which a part of blastocyst has started to differentiate into trophoblasts. Using a highly specific and sensitive ELISA, no sHLA-G protein was detectable in any sample, despite the fact that 27 of the 109 samples were from successfully implanted embryos. These results indicate that 2-6-day embryos do not secrete sHLA-G detectable by ELISA, and therefore that sHLA-G in culture medium is not a useful for successful implantation at this stage of development.

The involvement of HLA-E and -F in pregnancy.

The human MHC class I genes, HLA-E, -F and -G are referred to as non-classical or class Ib genes and are distinguished from their close relatives (the classical HLA class I genes) by expression patterns and low allelic polymorphism. To date, most studies that relate these molecules to the immunology of pregnancy have concerned only HLA-G. However, recent advances have suggested potential unique roles as well for HLA-E and HLA-F in pregnancy. A notable advance was the observation that all three proteins are expressed on the surface of extravillous trophoblast that has invaded the maternal decidua. Given this expression site, possibly the only cell type in human development where this occurs, it is logical to hypothesize that all three antigens, each with its own unique receptor-ligand interaction(s), contribute collectively to enable the growth of the developing child. In this review, we examine and discuss the accumulated data on expression and function of HLA-E and HLA-F and attempt to relate what is known to the involvement of HLA-E and -F in human pregnancy.

The surface expression of HLA-F on decidual trophoblasts increases from mid to term gestation.

HLA-F has recently only begun to be studied in earnest, and has been thought not to be expressed on the cell surface. However, in our previous report, we demonstrated surface expression of HLA-F on extravillous trophoblasts (EVTs) invading the decidua in term placental tissues. To better understand its function, we attempted to determine when surface expression of HLA-F begins during normal pregnancy, and whether there is a difference in expression between normal and preeclamptic placentas, by comparing the expression of HLA-G and -E by immunohistochemical staining with anti-HLA-E, -F and -G antibodies (3D12, 3D11 and 87G, respectively). In EVTs, HLA-F was expressed only in the cytoplasm weakly during the first trimester, after which expression increased and moved to the cell surface with the progression of pregnancy from the second trimester, which was confirmed by the results of double-labeled immunofluorescence staining with anti-HLA-F and anti-HLA-G antibodies, and by flow cytometry using trophoblasts isolated from the decidua. HLA-E showed similar expression as HLA-F, though it was expressed on the cell surface from the first trimester, while HLA-G was expressed strongly in the cytoplasm and on the cell surface during all stages of pregnancy. The expressions of HLA-E, -F and -G in preeclamptic placentas were not different from those in normal placentas, though there were a greater number of necrotic EVTs in preeclampsia. The increase in expression of HLA-E and HLA-F from the second trimester to full term was coincident with the timing of rapid growth of the fetus. Our results suggest that these may function together to prepare an environment that supports fetal growth.

HLA-F and MHC-I Open Conformers Bind Natural Killer Cell Ig-Like Receptor KIR3DS1.

Based on previous findings supporting HLA-F as a ligand for KIR3DL2 and KIR2DS4, we investigated the potential for MHC-I open conformers (OCs) as ligands for KIR3DS1 and KIR3DL1 through interactions measured by surface plasmon resonance. These measurements showed physical binding of KIR3DS1 but not KIR3DL1 with HLA-F and other MHC-I OC while also confirming the allotype specific binding of KIR3DL1 with MHC-I peptide complex. Concordant results were obtained with biochemical pull-down from cell lines and biochemical heterodimerization experiments with recombinant proteins. In addition, surface binding of HLA-F and KIR3DS1 to native and activated NK and T cells was coincident with specific expression of the putative ligand or receptor. A functional response of KIR3DS1 was indicated by increased granule exocytosis in activated cells incubated with HLA-F bound to surfaces. The data extend a model for interaction between MHC-I open conformers and activating KIR receptors expressed during an inflammatory response, potentially contributing to communication between the innate and adaptive immune response.

sHLA-G and sHLA-I levels in follicular fluid are not associated with successful implantation.

In the field of in vitro fertilization (IVF), useful markers for the prediction of successful implantation for oocyte or embryo selection are essential. It has been reported that sHLA-G (sHLA-G1/HLA-G5) could be detected in the supernatant of the fertilized embryo and in follicular fluid samples (FFs), and that the presence of sHLA-G was related to successful implantation. If sHLA-G could be used as a marker of oocyte selection from multiple FFs, oocytes could be selected without physical contact, thus reducing the likelihood of damage. To investigate the potential for sHLA-G as a marker of oocyte selection from multiple FFs in one patient, protein levels of total protein, sHLA-G, and sHLA-I (sHLA-A, B, and C) were examined in FFs. The variation among multiple FFs in total protein level and sHLA-G level was not related to successful pregnancy. The average sHLA-I levels did not differ in the successful implantation and unsuccessful implantation groups, indicating that sHLA-I levels were not related to successful pregnancy. Furthermore, sHLA-G in FFs was not detected by western blotting, despite being detected by ELISA, while sHLA-I was detected by both ELISA and western blot. These data suggest that sHLA-G in FF might not be a useful marker for oocyte selection as measurements of sHLA-G were inconsistent and there was no association with successful pregnancy. Further, more rigorously tested ELISA systems for detecting sHLA-G in body fluids are necessary before the utility of sHLA-G for diagnosis can be established.

An integrated genotyping approach for HLA and other complex genetic systems.

Clinical immunogenetics laboratories performing routine sequencing of human leukocyte antigen (HLA) genes in support of hematopoietic cell transplantation are motivated to upgrade to next-generation sequencing (NGS) technology by its potential for cost savings as well as testing accuracy and flexibility. While NGS machines are available and simple to operate, there are few systems available that provide comprehensive sample preparation and data analysis workflows to complete the process. We report on the development and testing of the Integrated Genotyping System (IGS), which has been designed to specifically address the challenges associated with the adoption of NGS in clinical laboratories. To validate the system for a variety of sample DNA sources, we have tested 336 DNA specimens from whole blood, dried blood spots, buccal swabs, and lymphoblastoid cell lines. HLA class I and class II genotypes were derived from amplicon sequencing of HLA-A, -B, -C for exons 1-7 and HLA-DPA1, -DPB1, -DQA1, -DQB1, -DRB1, -DRB3, -DRB4, -DRB5 for exons 1-4. Additionally, to demonstrate the extensibility of the IGS to other genetic loci, KIR haplotyping of 93 samples was carried out in parallel with HLA typing using a workflow based on the HLA system. These results are discussed with respect to their applications in the clinical setting and consequent potential for advancing precision medicine.

Asphyxial death related to postextraction hematoma in an elderly man.

We here report an autopsy case of a man in his seventies who died from asphyxia due to compression of the trachea caused by postextraction bleeding after extraction of his left mandibular third molar by a dentist in private practice. On the morning after the tooth extraction, he had complained of dyspnea and became unconscious at home. Although he was brought to the emergency room by ambulance, he died 7 days later without regaining consciousness. Autopsy examination revealed that the lingual side of the alveolar bone was fractured at the extraction socket. Moreover, subcutaneous bleeding that extended from the extraction socket to the thyrohyoid ligament in the cervical region and deviation of the epiglottis due to the bleeding were observed. Histological findings revealed liver cirrhosis; there were no significant findings in other organs. On the basis of these findings, we concluded that alveolar bone fracture occurred during the extraction and that the bleeding spread to the cervical region. Thus, the patient had died from asphyxia resulting from airway obstruction caused by cervical subcutaneous bleeding derived from postextraction bleeding. We emphasize that tooth extraction may cause fatal complications in patients with bleeding tendencies, particularly in the elderly.

Soluble MICB serum levels correlate with disease stage and survival rate in patients with oral squamous cell carcinoma.

BACKGROUND: Expression of ligands of natural killer group 2D (NKG2D) immunoreceptors, such as major histocompatibility complex class I-related chain A/B (MICA/B), has been proposed to play an important role in tumour immunosurveillance. Soluble forms of MICA/B are increased in sera of cancer patients and are postulated to impair antitumour immune response by downregulating expression of NKG2D immunoreceptors. Serum levels of soluble MICA have been shown to be of diagnostic significance in malignant diseases. AIMS: The potential of soluble MICB (sMICB) as a marker for oral squamous cell carcinoma (OSCC) was investigated. RESULTS: sMICB levels did not differ significantly from those in normal control individuals. However, the findings indicate that sMICB levels are significantly increased in stage IV OSCC and high sMICB levels are significantly associated with decreased survival rates in patients.

An association between the MICA-A5.1 allele and an increased susceptibility to oral squamous cell carcinoma in Japanese patients.

BACKGROUND: Recently, a new polymorphic gene family called the major histocompatibility complex class I chain-related gene A (MICA) was discovered about 40 kb centromeric to HLA-B gene. The MICA protein, expressed on epithelial cells and many kinds of tumor cells, serves to regulate immune function. The MICA protein is thought to activate immune function on mucosal tissue by binding to NKG2D which is expressed on most natural killer cells, CD8 positive T cells, and gamma delta T cells. An association between MICA gene polymorphisms and the development of oral squamous cell carcinoma (OSCC) has also been reported. OBJECTIVE: This study was designed to test this association in Japanese patients with OSCC. METHODS: The (GCT)(n) polymorphisms of the MICA gene was investigated in 123 patients with OSCC and 188 normal controls using polymerase chain reaction amplification and denaturing polyacrylamide gel electrophoresis. RESULTS: Five alleles, namely A4, A5, A6, A9, and A5.1, were found in both groups. The phenotype frequency of the MICA-A5.1 allele was significantly higher in patients with OSCC when compared with normal controls (OR 1.707, 95% CI 0.76-3.45, P=0.042). Also, the microsatellite frequency of the MICA-A5.1 allele was significantly higher in patients with OSCC compared with normal controls (OR 1.664, 95% CI 0.82-3.42, P=0.021). Lastly, the frequency of the MICA-A5.1 allele was significantly higher in those with lymph node metastasis from OSCC compared with normal controls (OR 2.605, 95% CI 1.14-5.27, P=0.026). CONCLUSIONS: These results suggest that the MICA-A5.1 allele may be associated with an increased susceptibility to OSCC in Japan.

Inhibition of PAR4 signaling mediates ethanol-induced attenuation of platelet function in vitro.

BACKGROUND: Reduction in coronary heart disease morbidity in response to moderate consumption of alcoholic beverages may be partly mediated by ethanol-induced inhibition of platelet function. However, the precise mechanisms by which ethanol modulates platelet activation induced by thrombin, which plays a central role in hemostasis, remain unclear. The goal of this study was to investigate ethanol-induced changes in platelet function and clarify the underlying mechanisms including PAR1 and PAR4 activity and [Ca2+]i dynamics in vitro. METHODS: Platelet aggregation, increase in intracellular calcium ([Ca2+]i), and release of platelet factor 4 and beta-thromboglobulin induced by alpha-thrombin, PAR1-agonist peptide (AP), or PAR4-AP were assessed in the presence or absence of ethanol. RESULTS: Ethanol exposure inhibited low-dose thrombin (0.5 nM)-induced aggregation but not an increase in [Ca2+]i. In contrast, ethanol had no effect on high-dose thrombin (10 nM)-induced aggregation or the [Ca2+]i increase. Ethanol did not significantly inhibit thrombin-induced release of platelet factor 4 and beta-thromboglobulin. Ethanol reduced PAR1-AP-induced aggregation, but did not affect the spike form of [Ca2+]i increase. In contrast, ethanol inhibited the increase in [Ca2+]i as well as the aggregation in response to PAR4-AP and resulted in delayed [Ca2+]i peak time. Furthermore, ethanol inhibited both PAR1-AP- and PAR4-AP-induced platelet factor 4 and beta-thromboglobulin release. CONCLUSIONS: These data suggest that ethanol inhibits platelet aggregation via inhibition of PAR4 signaling and subsequent inhibition of Ca2+ influx and granule release. This phenomenon may contribute to the reduction in coronary heart disease morbidity in response to consumption of alcoholic beverages.

HLA-E, HLA-F, and HLA-G polymorphism: genomic sequence defines haplotype structure and variation spanning the nonclassical class I genes.

Despite several studies that defined the polymorphism of the nonclassical human leukocyte antigen-E (HLA-E), HLA-F, and HLA-G genes, most polymorphisms thus far examined in correlative studies were derived from the coding sequences of these genes. In addition, some discrepancies and ambiguities in the available data have persisted in current databases. To expand the data available and to resolve some of the discrepant data, we have defined protocols that allow for the amplification of 6 to 7 kb of contiguous genomic sequence for each gene, including all of the coding and intron sequences, approximately 2 kb of 5' flanking promoter sequence, and 1 kb of 3' flanking sequence. Using long-range polymerase chain reaction (PCR) protocols, generating either one or two PCR products depending on the locus, amplified genomic DNA was directly sequenced to completion using a set of about 30 primers over each locus to yield contiguous sequence data from both strands. Using this approach, we sequenced 33 genomic DNAs, from Asian, African American, and Caucasian samples. The results of this analysis confirmed several previously reported coding sequence variants, identified several new allelic variants, and also defined extensive variation in intron and flanking sequences. It was possible to construct haplotype maps and to identify tagging single nucleotide polymorphisms that can be used to detect the composite variation spanning all three genes.

Genetics of the immune response: identifying immune variation within the MHC and throughout the genome.

With the advent of modern genomic sequencing technology the ability to obtain new sequence data and to acquire allelic polymorphism data from a broad range of samples has become routine. In this regard, our investigations have started with the most polymorphic of genetic regions fundamental to the immune response in the major histocompatibility complex (MHC). Starting with the completed human MHC genomic sequence, we have developed a resource of methods and information that provide ready access to a large portion of human and nonhuman primate MHCs. This resource consists of a set of primer pairs or amplicons that can be used to isolate about 15% of the 4.0 Mb MHC. Essentially similar studies are now being carried out on a set of immune response loci to broaden the usefulness of the data and tools developed. A panel of 100 genes involved in the immune response have been targeted for single nucleotide polymorphism (SNP) discovery efforts that will analyze 120 Mb of sequence data for the presence of immune-related SNPs. The SNP data provided from the MHC and from the immune response panel has been adapted for use in studies of evolution, MHC disease associations, and clinical transplantation.

Necrotic feature of the trophoblasts lacking HLA-G expression in normal and pre-eclamptic placentas.

PROBLEM: Human leukocyte antigen (HLA)-G is thought to be expressed in all placental extravillous trophoblasts (EXTs). In pre-eclamptic placentas, a lack of HLA-G expression on EXTs had been found, and deduced as a possible cause of pre-eclampsia. However, a subset of EXTs lacking expression of HLA-G can also be found in normal placenta. Therefore, we sought to compare these cells in normal and pre-eclamptic placentas. METHODS OF STUDY: Frozen sections of normal and pre-eclamptic placentas were examined by immunohistochemical staining using HLA-G monoclonal antibody 87G, histochemical enzymatic analysis of succinate dehydrogenase (SDH) and ultrastructural analysis. RESULTS: A subset of EXTs lacking HLA-G expression was found in both normal and pre-eclamptic placentas. These cells showed necrotic features such as the swelling of cells, eosin-achromatophilia, the loss of SDH activity and swelling mitochondria. Cells from both tissues were identical with regard to these features. CONCLUSION: The features of the EXTs lacking HLA-G expression indicated they had undergone necrosis and thus could not express HLA-G protein. Therefore, an alternative interpretation to the lack of HLA-G expression in pre-eclamptic placentas is that it is the result of cell death and not the cause.

Protein expression and peptide binding suggest unique and interacting functional roles for HLA-E, F, and G in maternal-placental immune recognition.

In this study we focused on the structure and expression of the HLA-E, F, and G class I complexes in placental tissue. Structural analysis included an examination of the peptides bound to soluble and membrane forms of the HLA-G complex isolated directly from placenta. An important distinction was observed from HLA-G bound peptides previously isolated from transfectant cells. Thus, the number of distinct moieties bound to placental-derived proteins was substantially lower than that bound to transfectant-derived HLA-G. Indeed, a single peptide species derived from a cytokine-related protein alone accounted for 15% of the molar ratio of HLA-G bound peptide. To further examine HLA-E and its potential to bind peptide, notably that derived from HLA-G, we combined new Abs to examine expression in placental tissues for all the known forms of the nonclassical class I molecules. Whereas membrane HLA-G was found in extravillous trophoblasts, soluble HLA-G was found in all placental trophoblasts, including villous cytotrophoblasts and syncitiotrophoblasts. Further, HLA-E was found in all cells that expressed either form of HLA-G, consistent with HLA-E being complexed with the HLA-G signal sequence-derived nonamer in these cells. Finally, using new reagents specific for HLA-F, a restricted pattern of expression was observed, primarily on extravillous trophoblasts that had invaded the maternal decidua. Comparative staining indicated that HLA-F was on the surface of these cells, defining them as the first to demonstrate surface expression of this Ag and the first cell type identified to express all three nonclassical HLA class I Ags simultaneously.