Specific HLA-A Research Articles

Expression of transporter associated with antigen processing 1 and 2 (TAP1/2) in malignant melanoma cell lines.

TAP1 and TAP2 molecules are involved in the transport of peptides prior to their association with class I molecules and are mandatory for efficient antigen presentation. To investigate whether loss of expression of TAP1 or TAP2 is a likely mechanism of immune escape in malignant melanoma, TAP1 and TAP2 mRNA was analyzed by RT-PCR in 39 melanoma cell lines expressing at least 2 of the known melanoma-associated antigens, tyrosinase, Melan-A/MART-1, gp100, MAGE-1 and MAGE-3. All 39 cell lines expressed both TAP1 and TAP2 at the mRNA level. To investigate other factors potentially involved in immune escape, the expression of LMP2, LMP7, HLA class I molecules, beta2-microglobulin (beta2m) and specific HLA-A alleles was evaluated by RT-PCR and FACS analyses. All 39 cell lines expressed LMP2, LMP7 and beta2m. A single cell line (FM37) had lost the expression of class I molecules, and this same cell line showed loss of expression of the HLA-A2 heavy chain. No cell lines showed loss of expression of the HLA-A1 heavy chain. Based on our studies of in vitro established cell lines, loss of TAP1/2 or LMP2/7 expression does not appear to be a common mechanism of immune escape in malignant melanoma.

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.

Complete subtyping of the HLA-A locus by sequence-specific amplification followed by direct sequencing or single-strand conformation polymorphism analysis.

A variety of reasons related to the HLA class I system has complicated the application of molecular approaches to HLA class I typing. Here we present a PCR-based HLA-A typing strategy considering the sequence variations of the two most polymorphic exons which allows complete subtyping of the HLA-A locus. The method is based on a sequence-specific amplification identifying the serologically defined HLA-A specificities. The PCR products generated by these group-specific primers bear the sequence information necessary for a postamplification specificity step. The primer pairs are located within one exon, either exon 2 or exon 3, which avoids amplification of polymorphic intron sequences allowing subsequent single-strand conformation polymorphism analysis and facilitating direct sequencing. Using this method we investigated 48 cell lines and 153 clinical samples. 23 PCR reactions are performed per individual for the assignment of the serological specificities A1-A80. The reproducibility was 100% in all cell lines and 85 clinical samples typed on two separate occasions. With the exception of 13 out of 231 possible serological combinations all homozygous and heterozygous combinations of A1-A80 can be distinguished by specific amplification patterns. Comparing the PCR based typing results with those of serology in 12% a discrepancy was found. Solid-phase sequencing or SSCP analysis of the group-specific PCR fragments allowed complete subtyping of the HLA-A locus. This strategy can identify all 48 HLA-A alleles based on the sequence variations of the 2nd and 3rd exon. 1128 homozygous and heterozygous allele combinations are possible for the HLA-A locus. Only 4 out of these 1128 allele combinations remained unresolved.

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 fibroblast 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.

Genetic predetermination of quantitative expression of HLA antigens in platelets and mononuclear leukocytes

In view of the potential functional importance of quantitative expression of HLA antigens, a series of studies were conducted to determine the relative quantities of specific HLA-A and -B antigens expressed in MNLs and platelets of HLA-phenotyped family members and unrelated individuals. An mAb that reacts with a well-defined monomorphic epitope in the α 3 domain of the heavy chains of HLA molecules was developed and used to quantify each HLA-A or -B antigen on western blots of IEF gels. The results of these studies demonstrated that the relative quantities of HLA-A and -B antigens in platelets and MNLs of an individual did not change over time. Further studies showed that the relative quantities of HLA-A and -B antigens for haplotypes shared among the first-degree relatives were always the same and followed Mendelian inheritance. In contrast, the relative quantities of HLA-A and -B antigens for a haplotype shared by unrelated individuals varied significantly. All these findings support the hypothesis that the quantitative expression of HLA antigens is genetically predetermined and may play important roles in determining disease susceptibility and severity. Human Immunology 38, 243–250 (1993)

Structure of the HLA-A*0204 antigen, found in South American Indians. Spatial clustering of HLA-A2 subtype polymorphism

The primary structure of the HLA-A2 subtype A*0204 (isoelectric focusing variant A2.4) has been determined. cDNA encoding this subtype was amplified by the polymerase chain reaction. Four independent full-length cDNA clones encoding A*0204 were analyzed to obtain a consensus sequence for this subtype. A*0204 differs from A*0201 by a single nucleotide change of G to T through the coding regions, resulting in an Arg to Met change at position 97. This substitution accounts for the isoelectric focusing pattern of the subtype. The same change occurs in other HLA-A specificities in association with other changes in its vicinity. The absence of additional substitutions in A*0204 suggests that it could have arisen from A*0201 by point mutation, and that recurrent mutations may take place during HLA diversification. The spatial location of this change implies that A*0204 must be a functional variant. Comparison of its sequence with other HLA-A2 subtypes reveals that much of the HLA-A2 subtype polymorphism is generated by variations in four neighboring positions, including position 97, which are located in two adjacent beta-strands on the floor of the peptide binding site of the molecule.

Evaluation of individual specificities of class I HLA on platelets by a newly developed monoclonal antibody

In order to quantify each specific HLA-A or-B antigen on platelets, a monoclonal antibody against HLA heavy chains was developed and designated as 2F2 monoclonal antibody. This monoclonal antibody reacted on Western blot with platelet HLA from each 10 individuals with different HLA phenotypes and precipitated all 35S-methionine-labeled HLA-A and -B antigens from three different Epstein-Barr Virus-transformed lymphoblastoid cell lines. The results indicate that the 2F2 monoclonal antibody recognizes an epitope shared by different HLA-A and -B antigens. The quantitative variation of specific HLA antigens on platelets was then studied in nine different donors by isoelectric-focusing gel electrophoresis and immunoblot using the 2F2 monoclonal antibody. The results of our studies showed that the shared HLA antigens such as A2, B35, and B62, varied three- to fivefold among different individuals and individual HLA-A or -B antigen was not equally expressed on a person's platelets. The relative quantities of specific HLA-A and -B antigens on lymphocytes were also noted to be the same as those on platelets. The finding suggests that differential expression of HLA specificities may not be restricted to platelets but is a more general phenomenon including other nucleated cells.

A sensitive and specific ELISA using monoclonal capture antibodies for the detection of HLA antigens in blood stains

A double antibody ELISA technique is described to detect HLA antigens in extracts of blood stains. The assay involves capture of free HLA determinants using an immobilized monoclonal antibody directed against monomorphic regions of HLA class I and HLA class II antigens. The captured antigens are then detected using alloantisera directed against the polymorphic regions of the captured HLA entities. The technique is able to detect specific HLA-A, B, and DR antigens in extracts prepared from blood smears as well as from dried and freshly thawed lymphocytes. The assay may be of potential use in forensic medicine, particularly in instances where extraction of nucleic acids for finger printing is not feasible.

The distribution of HLA-A, B, C, DR antigens in Chinese patients with hepatocellular carcinoma in Taiwan.

To evaluate the role of genetic factor in hepatocellular carcinoma, HLA-A, B, C and DR locus antigens were typed in 170 Chinese patients. Forty-three HLA-A, B, C, DR-related antigens were assayed by the conventional method. No significant risk antigen can be identified among these antigens. The distribution of each antigen was not related to sex. HBsAg, and alpha-fetoprotein status. The results suggest that there are no specific HLA-A, B, C and DR antigens patterns or frequencies associated with the development of hepatocellular carcinoma.

Isolation and characterization of monoclonal mouse cytotoxic T lymphocytes with specificity for HLA-A,B or -DR alloantigens.

Isolation and characterization of monoclonal mouse cytotoxic T lymphocytes with specificity for HLA-A,B or -DR alloantigens.

HLA phenotypes in Mexican Americans with tuberculosis.

HLA-A, -B, and -C phenotype distributions in 100 Mexican Americans with active tuberculosis were compared with HLA phenotypes in 50 healthy, tuberculin-skin-test-positive and 50 healthy, tuberculin-skin-test-negative Mexican Americans. Although there existed differences in the phenotype frequencies of 5 antigens (Aw30, Aw33, B7, B15, and B17) among the 3 study groups, these differences were not significant using p values that were corrected for the number of antigens tested. Thus, susceptibility (or resistance) to tuberculosis in Mexican Americans does not appear to be linked to a specific HLA-A, -B, or -C phenotype.

HLA Linkage vs Association in Hypertrophic Cardiomyopathy: Evidence for the Absence of an Association in a Heterogeneous Caucasian Population

Human leukocyte antigen (HLA) tissue-typing studies on patients with genetically transmitted hypertrophic cardiomyopathy have demonstrated an HLA linkage in a Caucasian and black patient group and an HLA-DR locus association in a Japanese population sample. To confirm whether a specific HLA antigen(s) might serve as a marker for hypertrophic cardiomyopathy, we performed tissue-typing studies on 50 unrelated, normotensive North American Caucasians with the disorder. Patients were subdivided into three hemodynamic subgroups: obstructive (35), provocable (ten), and nonobstructive (five). Although there was an increased frequency of the B12 and AW32 HLA antigens in the total group, after correction of P values for the number of antigens studied, the associations were not statistically significant. Analysis of the HLA antigen frequencies in the three hemodynamic subgroups yielded no statistically significant HLA-A, B, or C locus associations, and no unusual deviation in linkage disequilibrium between A and B locus antigens was observed. We conclude that although on the sixth chromosome there may be a susceptibility gene for hypertrophic cardiomyopathy, which segregates with a specific haplotype in a given family, no specific HLA-A, B, or C locus antigen was found useful as a marker. HLA-DR locus antigen typing might prove useful in this population.

HLA-A, -B, and -DR specificities on human monocytes.

Monocyte-enriched cell suspensions obtained by a plate adherence method from 57 blood donors were typed by microcytotoxicity for HLA-A, -B and -DR determinants. Parallel assays were performed with autologous unfractionated lymphocytes for HLA-A and -B determinants and with autologous B-lymphocytes for HLA-DR determinants. Correlation coefficients (r values) were calculated for nine HLA-A specificities (r greater than 0.79), 16 HLA-B specificities (r greater than 0.56) and seven HLA-DR specificities (r greater than 0.81). For certain HLA-A and -B specificities detection was less readily achieved on monocytes than on autologous lymphocytes. Extra reactions were observed in some instances. With sera defining HLA-DR specificities, monocytes and B-cells showed reactivity of almost identical strength and reliability.

Characterization, evolution, and molecular basis of a polymorphic antigenic determinant shared by HLA-A and B products

In our search for monoclonal antibodies defining specific HLA-A,B antigens we have found a significant number of antibodies with polymorphic reactions that are not explicable with our current picture of HLA-A,B interrelationships. Analysis of one antibody (MB40.1) in some detail shows that it recognizes subsets of both A and B antigens and that it is able to split highly cross-reactive pairs of A or B antigens. These results address an outstanding difference in the serological interpretation of H-2, where these types of public reactions have been well established, from HLA, where they have been poorly characterized. Comparison of these types of serological reactions with amino acid sequence data and the apparent evolutionary stability of these broad cross-reacting determinants suggests a simple model for their molecular nature based upon limited amino acid variability and convergent evolution.

Fab2 fragments from HLA xenoantisera specifically block cytolysis mediated by HLA-AB alloantisera.

Fab2 fragments from antisera raised in rabbits with partially purified cellular and serum HLA antigens were tested for their ability to block the cytolytic activity of operationally specific HLA-A,B alloantisera. One Fab2 fragment preparation blocked the cytolytic activity of all the HLA-A,B alloantisera tested; the remaining nine inhibited the lytic activity of alloantisera to certain HLA-A,B allospecificities, suggesting that these xenoantisera contain antibody to certain HLA-A,B allotype determinants or to closely associated structures. In contrast to previous reports in the literature none of the xenoantisera contained significant amounts of antibodies to human beta 2-microglobulin.

INFLUENCE OF PREVIOUS IMMUNIZATION ON SKIN GRAFT SURVIVAL

The results of skin grafts transplanted in immunized and nonimmunized recipients was analysed. Specific sensitization for HLA-A or B determinants shortens graft survival if the recipients were immunized by s.c. injections of leukocytes. When the recipients had been pregnant, no such influence of specific HLA-A or B sensitization could be demonstrated. The variance in mean survival times of grafts exchanged between mixed lymphocyte culture (MLC)-positive donor-recipient combinations was significantly smaller than the variance in mean survival time (MST) of grafts exchanged between MLC-negative combinations. This difference could be the result of the influence of allograft immune-activating determinants of different strength in the MLC-negative donor-recipient combinations. Also the variance in MST of grafts in immunized recipients was significantly larger than the variance in MST of grafts in nonimmunized recipients. Apart from the obvious effect of HLA-A and B sensitization, other less well documented factors must have influenced graft survival. We did not find evidence for a graft enhancing effect of B cell-specific antibodies.