High frequency of HLA-A*0207 subtype in Chinese population.

BACKGROUND: HLA-A2 is the most polymorphic and most common HLA phenotype found in various ethnic populations. Seventeen HLA-A2 subtypes have been reported and characterized by molecular techniques. Differences among these subtypes are limited to a few amino acids. Among them, HLA-A*0201 is the predominant subtype among whites. The results of a recent study, however, suggest that the HLA-A*0207 subtype may be present at a high frequency in the Chinese population. STUDY DESIGN AND METHODS: To determine the exact frequency of the HLA-A*0207 subtype in the Chinese population, genomic DNA samples obtained from 54 HLA-A2-positive Chinese in Taiwan were studied by using sequence-specific primers and polymerase chain reaction. RESULTS: HLA-A*0207 was present in 56 percent of the studied subjects. The estimated gene frequency for HLA-A*0207 is 17.8 percent in the Chinese population. CONCLUSION: HLA-A*0207 is the most common HLA-A2 subtype among Chinese. The high frequency of the HLA-A*0207 allele in this population offers a unique opportunity to study the ways in which different HLA-A2 subtypes may influence the clinical outcome of allograft transplantation and the disease susceptibility of recipients.

Functional significance of varied quantitative and qualitative expression of HLA-A2.1 antigens in determining the susceptibility of cells to cytotoxic T lymphocytes.

To determine whether varied quantitative HLA expression affects the susceptibility of target cells to CTLs, a panel of 15 EBV-transformed lymphoblastoid cell lines expressing a fivefold difference of surface HLA-A2.1 antigens were employed. The susceptibility of these cell lines to HLA-A2.1-restricted and influenza virus matrix peptide-specific CTLs was correlated with the amounts of HLA-A2.1 antigens expressed on their surface. The results show a linear correlation between both parameters using exogenous viral peptide. The same linear correlation was observed when target cells infected with influenza virus were studied. These findings support the hypothesis that the amount of HLA antigens expressed on the cell surface is functionally significant in determining the susceptibility of target cells to CTLs. During our study, we also found that two HLA-A-2.1-positive cell lines were unresponsive to the CTL. Further investigation of the amino acid sequences of these cell lines reveals that their HLA-A2.1 antigens belong to the HLA-A0207 subtype which is different from HLA-A0201(A2.1) by one nucleotide. This difference results in an amino acid substitution from tyrosine to cysteine at position 99 of HLA-A2.1 heavy chains. Using a peptide-induced reconstitution assay, it was shown that failure of the peptide binding is responsible for the absence of cytotoxicity. This finding supports the hypothesis that amino acid 99 plays an important role in determining the peptide-binding specificity of HLA-A2 molecules.

A combined approach for quantitation of each specific HLA-A or -B antigen expressed on cells.

In order to measure the amount of each individual HLA-A or -B antigen expressed in or on a cell without relying on monoclonal antibodies to different specific HLA antigens, we have developed a combined approach that consists of two separate measurements. The first measurement is to determine the relative quantities of different HLA-A and -B antigens in lysates of whole cells using IEF gel electrophoresis, immunoblotting with 171.4 anti-HLA heavy chain monoclonal antibody, and scanning densitometry. The second measurement is to determine the concentration of total HLA antigens expressed in or on a cell using an enzyme-linked immunoassay or a surface binding assay based on W6/32 anti-HLA monoclonal antibody. The quantity of each specific HLA antigen in or on a cell then is calculated from the results of these two measurements. To validate this combined approach, we conducted studies to show that the amounts of different specific HLA antigens measured by this approach were linearly correlated with those measured by FITC-labeled monoclonal antibodies and immunofluorescence flow cytometry in platelets and lymphoblastoid cell lines. We also demonstrated that the relative quantities of different HLA-A and -B antigens determined from the lysates of whole cells were the same as those expressed on the cell surface. These findings indicated that the newly developed combined approach can be applied to quantify each specific HLA-A or -B antigen expressed in or on a cell.

Proportional amplification of individual HLA-A and -B antigens during upregulated expression of total class I HLA molecules.

Our recent studies demonstrated that each specific HLA-A or -B antigen is not expressed in equal quantity in cells of an individual and that the relative amounts of different HLA-A and -B antigens are genetically predetermined following Mendelian laws. These findings suggest the potential genetic importance of varied quantitative HLA expression on target cells in determining the sensitivity to cytotoxic T lymphocytes. It would be important to know whether the amounts of different HLA antigens are differentially or proportionally amplified after upregulated expression of total HLA antigens. We have therefore determined the effects of IFN treatment, EBV transformation, and influenza virus infection on the quantitative expression of total HLA antigens and the relative quantities of different specific HLA-A and -B antigens in human fibroblasts cell line and peripheral blood mononuclear leukocytes. In contrast to earlier studies using the transfected HLA genes, our results show that different individual HLA-A and -B antigens are proportionally and not differentially amplified during upregulated expression of total class I HLA molecules. This finding indicates that the genetic predetermination of varied quantitative expression of HLA antigens may play a role in influencing antiviral immunity and disease susceptibility.

Quantitation and characterization of plasma HLA in neonates of different gestational ages.

The functional significance of plasma HLA class I antigens is unclear. They are thought to have an immunomodulatory role and be tolerogenic in transplant settings including the materno-fetal semi-allograft. There is, however, no available data on the concentrations of soluble HLA class I antigens in fetuses or newborns. We therefore determined plasma HLA class I antigen levels in 93 neonates born at different gestational ages and compared them to those in 66 healthy adults. The mean plasma HLA concentration in cord blood obtained from these neonates (0.30 +/- 0.15 microgram/ml, mean +/- SD) was significantly lower (p < 0.0001) than in the adults (0.77 +/- 0.44 microgram/ml). No correlation between the plasma HLA levels and the gestational ages of the neonates was detected. Characterizing the plasma HLA class I antigens by immunoprecipitation and immunoblotting, four different molecular weight forms, 44, 39, 36 and 34 kDa, were recognized. Their distribution in neonates was not different from that in adults. Since the circulating leukocytes are a probable source of plasma HLA class I antigens, we measured the surface HLA expression on leukocytes in 4 neonates and 4 adults by immunofluorescent flow cytometry. The fluorescence intensities on neonatal granulocytes and lymphocytes were 50% of those on corresponding adult cells. This finding suggests that the reduced HLA expression by neonatal leukocytes may be partially responsible for the lower concentration of HLA class I antigens in neonatal plasma.

Insulin-dependent diabetes in the NOD mouse model. I. Detection and characterization of autoantibody bound to the surface of pancreatic beta cells prior to development of the insulitis lesion in prediabetic NOD mice.

Type I, insulin-dependent diabetes (IDD) results from an autoimmune response against the insulin producing pancreatic beta cells. This autoimmune reaction involves both humoral and cell-mediated factors; nevertheless, the relative role of each remains unresolved. Furthermore, while adoptive transfer experiments have provided evidence for the role of T cells in beta cell destruction, the specific events which initiate leukocyte migration into the islets (insulitis) are unknown. Earlier studies indicated that NOD pancreatic beta cells may bind small amounts of autoantibody. Because of the possible importance of an early humoral response to the initiation of insulitis and subsequent disease, we have investigated a number of aspects of this phenomenon to determine the nature and specificity of the early autoantibodies as well as the time at which autoantibody binds to beta cells. Results of this study demonstrate that NOD/Uf mice are sensitized to islet-cell associated antigens, including GAD, prior to the first appearance of insulitis; that a small percentage of the beta cells of NOD/Uf mice have autoantibody bound to their surface prior to insulitis; that sera collected from preinsulitis NOD/Uf mice contain autoantibodies which will bind to beta cells of both IDD-prone and IDD-resistant mice; and that the autoantibodies which bind pancreatic beta cells are predominantly IgM with lesser amounts of IgG and IgA. These findings suggest that, in the natural course of IDD, insulitis may develop in response to an initial autoantibody-mediated injury of beta cells.