Detection of HLA-A*24 null alleles by DNA typing methods

Abstract: A number of cases have been identified (seven unrelated individuals from the Northern Ireland bone marrow donor registry and two family groups) where an HLA-A*24 allele fails to express the normal HLA-A24 antigen. Family information has revealed common haplotypes with respect to each non-expressed allele indicating that the occurrence of these mutations has been a recent event. Two methods for the clinical typing of these alleles have been evaluated - PCR-SSOP and PCR-SSCP analysis.     

Two tyrosinase nonapeptides recognized on HLA-A2 melanomas by autologous cytolytic T lymphocytes

A number of cytolytic T lymphocyte (CTL) clones derived from several melanoma patients have been found to recognize a majority of melanomas from HLA-A2 patients. We have reported previously that two such CTL clones recognize a product of the tyrosinase gene that is presented by HLA-A2. Here we show that one of these CTL clones recognizes a peptide encoded by the first nine amino acids of the putative signal sequence of tyrosinase. The other CTL clone recognizes a different tyrosinase peptide corresponding to amino acids 368–376. Both peptides contain consensus motifs of HLA-A2 binding peptides.     

HLA-A9 antibodies and epitopes

Fifty pregnancy alloantisera directed towards HLA-A23 and/or A24 antigens were investigated serologically in titration studies against the three sequenced HLA-A9 specificities, A23 (A*2301), A24 (A*2402) and A2403 (A*2403). The reaction patterns of the antisera fell into five categories which allowed the three HLA-A9 specificities to be easily differentiated. Based on the various titre cytotoxicity scores of the antisera five possible antibody specificities were defined: anti-A23; -A24; -A23/24; -A24/2403 and anti-A23/24/2403. One antiserum crossreacted with HLA-A1 and A24. Inspection of the amino acid sequences of 136 HLA-A, B and C molecules allowed the prediction of five unique epitopes corresponding to these antibody specificities, a possible epitope unique to A2403 and confirmation of a likely epitope shared by A1 and A24. These, together with the previously suggested epitopes HLA-A9/ A2/A28 and A1/A23/A24 together with the presence of Bw4 on the three HLA-A9 antigens suggests that the HLA-A9 family of antigens is characterized by a minimum of nine serologically definable epitopes.     

Identification of a novel gp100/pMel17 peptide presented by HLA-A*6801 and recognized on human melanoma by cytolytic T cell clones

Melanoma-associated peptides recognized by cytolytic T lymphocytes (CTL) in the context of several histocompatibility leukocyte antigens (HLA) are required for the development of specific immunotherapies. Using a transient transfection assay into COS-7 cells, we identified the gp100/pMel17 melanosomal protein as the shared antigen recognized by three independent CD8+ CTL clones in HLA-A*6801-restricted fashion. This finding was confirmed by the correlation between lack of gp100/pMel17 protein in a number of HLA-A*6801-positive melanomas and their resistance to lysis/cytokine production by the specific effectors. The gp100/pMel17 antigenic epitope was identified based on recognition of subfragments and on a computer-based prediction algorithm. Among a panel of gp100/pMel17-derived synthetic peptides only the 10-mer HTMEVTVYHR (gp100/pMel17182-191) induced tumor necrosis factor (TNF) release by CTL clones when pulsed on suitable target cells whereas both the 10-mer and the shorter 9-mer gp100/pMel17183-191 sensitized the same antigen-pulsed cells to lysis. In conclusion, the identification of the HTMEVTVYHR peptide will extend to HLA-A*6801 melanoma patients the possibility to exploit gp100/pMel17 melanosomal protein for experimental and clinical studies.     

Melanocyte lysis by cytotoxic T lymphocytes recognizing the MART-1 melanoma antigen in HLA-A2 patients with Vogt Koyanagi Harada disease

The MART-1/Melan-A melanoma antigen recognized by the majorityof HLA-A2-restricted tumorinfiltrating lymphocytes is a selfantigen expressed on melanocytes and the retina. We have investigatedwhether Vogt–Koyanagi–Harada (VKH) disease and sympatheticophthalmia (SO), systemic inflammatory disorders affecting variousorgans containing melanocytes, are autoimmune diseases directedtoward the MART-1 antigen. In two of three patients with VKHdisease and one patient with SO, CD8⁺ T cell clones (TCC) fromintraocular fluid of HLA-A2⁺ patients lysed T2 cells when pulsedwith a HLA-A2-binding MART-1 peptide, but not a HLA-A2-bindingpMel-17 or tyrosinase peptide, in a HLA-A2-restricted manner.These CD8⁺ TCC lysed both melanocytes and melanoma cells ina HLA-A2-restricted manner. In addition, CD8⁺ TCC recognizinga HLA-A2-binding MART-1 peptide were also established from peripheralblood mononuclear cells of a patient with VKH disease. In contrast,either CD4⁺ TCC from these patients or CD8⁺ TCC from the intraocularfluid of HLA-A2⁺ patients with uveitis associated with Behcet'sdisease or HTLV-I uveitis did not show this cytotoxicity. Theresults demonstrate that the MART-1 peptide-specific cytotoxicT lymphocytes lyse melanocytes in the eye of patients with VKHdisease or SO, suggesting that these diseases are autoimmunediseases directed toward the MART-1 antigen in HLA-A2⁺ patients.     

HLA-A*0201与EGFP融合蛋白表达载体的构建及其在K562细胞的表达和定位

目的:构建增强型绿色荧光蛋白(EGFP)与HLA-A^*0201(A^*0201)融合蛋白哺乳动物细胞表达载体,分析其在K562细胞中的表达和亚细胞定位。方法:以RT-PCR方法克隆A^*0201cDNA,构建A^*0201-EGFP融合蛋白表达载体,转染K562细胞,以流式细胞仪和激光共聚焦显微镜分析融合蛋白的表达及其亚细胞定位。结果:从两个HLA-A2阳性供者外周血细胞中克隆到A^*0201cDNA编码区全长序列。通过PCR方法在起始位点前加入Kozak序列并删除终止密码,成功构建A^*0201-EGFP融合蛋白表达载体。以该质粒转染K562细胞,5h后A^*0201和EGFP的表达百分率分别为25.12±2.26、27.37±3.59,24h后表达水平无明显提高。表达的融合蛋白主要分布于细胞膜上,胞内分布较少。相反,转染空载体的细胞不表达A^*0201分子,仅表达EGFP,且其在细胞内呈弥散样分布。结论:成功构建A^*0201-EGFP融合蛋白表达载体,并在K562细胞中得到表达,表达产物主要分布于细胞膜表面,提示表达该融合蛋白的K562细胞是潜在的人工抗原提呈细胞。     

Novel HLA-A*11 allele, A*1120, identified by sequence-based typing

In this report, we describe the identification of a human leucocyte antigen-A*11 (HLA-A*11) nucleotide sequence variant, a new HLA-A*1120 by using sequence-based typing (SBT). The new allele was detected during routine HLA typing by high-resolution SBT. Allele A*1120 showed one nucleotide difference with A*110101 at codon 152 (GCG-->GAG) resulting in an amino acid change from alanine to glutamate. Residue 152 is located on alpha(2)-helix of HLA class I molecule and involved in peptide binding by constructing E pocket of peptide-binding groove, implying that the change of the residue 152 would affect the binding affinity of peptides to A*1120 allele.     

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

Abstract: 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.     

HLA-A*02 allele frequencies and haplotypic associations in Koreans

We have investigated the frequencies of HLA-A*02 alleles and their haplotypic associations with HLA-B and -DRB1 loci in 439 healthy unrelated Koreans, including 214 parents from 107 families. All of the 227 samples (51.7%) typed as A2 by serology were analyzed for A*02 alleles using polymerase chain reaction (PCR)-low ionic strength-single-strand conformation polymorphism (LIS-SSCP) method. A total of six different A*02 alleles were detected (A*02 allele frequency 29.6%): A*0201/9 (16.6%), *0203 (0.5%), *0206 (9.3%), *0207 (3.0%), and one each case of *0210 and *02 undetermined type. Two characteristic haplotypes showing the strongest linkage disequilibrium were A*0203-B38-DRB]*1502 and A*0207-B46-DRB1*0803. Besides these strong associations, significant two-locus associations (P<0.001) were observed for A*0201 with B61, DRB1*0901 and DRB1*1401, and for A*0206 with B48 and B61. HLA haplotypes carrying HLA-A2 showed a variable distribution of A*02 alleles, and all of the eight most common A2-B-DR haplotypes occurring at frequencies of > or =1% were variably associated with two different A*02 alleles. These results demonstrate that substantial heterogeneity is present in the distribution of HLA-A*02 alleles and related haplotypes in Koreans.