Control of HIV-1 by an HLA-B*52:01-C*12:02 Protective Haplotype.

HLA-B*52:01-C*12:02, which is found in approximately 20% of all Japanese persons, is well known to be associated with ulcerative colitis and Takayasu arteritis. This haplotype is also known to be protective in individuals infected with human immunodeficiency virus (HIV) type 1. Recent studies showed that HLA-B*52:01-restricted HIV-1-specific T cells suppress HIV-1 and that HLA-C*12:02 together with KIR2DL2 play an important role in natural killer cell-mediated control of HIV-1. However, the role of HLA-C*12:02-restricted cytotoxic T lymphocytes (CTLs) in suppressing HIV-1 replication remains unknown. In the present study, we demonstrated that HLA-C*12:02-restricted CTLs specific for 2 immunodominant epitopes, Pol IY11 and Nef MY9, contributed to the suppression of HIV-1 replication in HIV-1-infected individuals. Further analysis demonstrated that these 2 HLA-C*12:02-restricted CTLs together with 4 HLA-B*52:01-restricted ones effectively suppressed HIV-1 in individuals with the HLA-B*52:01-C*12:02 haplotype. Thus, both HLA-C*12:02 and HLA-B*52:01 alleles contribute to HIV-1 suppression via both HIV-1-specific CTLs and natural killer cells in individuals with this haplotype.

Identification of a novel HLA-C*03 variant allele, C*03:280 by sequence-based typing.

The new allele, HLA-C*03:280 differs from C*03:04:01 by one nucleotide substitution at codon 35 (CGG→CAG).

Advancing allele group-specific amplification of the complete HLA-C gene--isolation of novel alleles from three allele groups (C*04, C*07 and C*08).

A variety of strategies have been designed for sequence-based HLA typing (SBT) and for the isolation of new human leucocyte antigen (HLA) alleles, but unambiguous characterization of complete genomic sequences remains a challenge. We recently reported a simple method for the group-specific amplification (GSA) and sequencing of a full-length C*04 genomic sequence in isolation from the accompanying allele. Here we build on this strategy and present homologous methods that enable the isolation of HLA-C alleles belonging to another two allele groups. Using this approach, which can be applied to sequence-based typing in some clinical settings, we have successfully characterized three novel HLA-C alleles (C*04:128, C*07:01:01:02, and C*08:62).

Next-generation HLA sequencing using the 454 GS FLX system.

Next-generation sequencing (NGS) of HLA class I and II loci (HLA-A, HLA-B, HLA-C, DRB1, DRB3, DRB4, DRB5, DQA1, DQB1, DPB1) is described here in detail using the 454 Life Sciences GS FLX System and Titanium chemistry. An overview of the protocol with our experience on sequence performance efficiencies, read depth and ambiguity analyses using the GS FLX System are also presented. A total of 14 HLA primer pairs with multiplex identifiers (MIDs) are used in clonal, amplicon-based pyrosequencing of up to 44 samples per plate using the GS FLX. Genotype assignment and ambiguity reduction -analysis is performed using Conexio Assign ATF 454 software. Clonal NGS gives a significant reduction in genotyping ambiguity during analysis of the highly complex HLA system.

HLA-C polymorphisms and PCR dropout in exons 2 and 3 of the Cw*0706 allele in sequence-based typing for unrelated Chinese marrow donors.

To evaluate the accuracy of SBT protocols for HLA-C and to better understand the HLA-C polymorphism in Chinese, 1795 unrelated CMDP donors were typed at exons 2, 3, and 4 of the HLA-C gene using the Atria commercial kit. Of the study subjects, 1768 showed conclusive typing results, whereas the other 27 showed inconclusive results. Subsequent full-length cloning and haplotype sequencing showed that 11 of the 27 inconclusive results could be explained by the presence of nine novel alleles identified: Cw*0130, 0624, 070206, 075602, 0766, 0767, 0820, 0821, and 0827. These novel alleles were generated by a total of 10 coding-region substitutions, eight of them being located in the antigen-binding groove. Cw*0766 and Cw*075602 were detected three and two times, respectively, in the 1795 donors. The other 16 inconclusive samples were retested by SBT using our in-house PCR primers; all of them were found to carry Cw*0706, which dropped out in exons 2 and 3 in the initial PCR using the commercial primers amplifying from 5' UTR to intron 3. Our results showed the importance of the full-length genomic sequence and intronic SNPs for the development of more accurate SBT. The allele distribution and novel alleles detected in this study also provide further insights into the HLA-C polymorphism in the Chinese Han population.

High frequency of altered HLA class I phenotypes in laryngeal carcinomas.

The exact frequency of HLA class I losses in human tumors is unknown. We have previously shown that primary breast and colorectal carcinomas frequently lose HLA class I molecules (88% and 73%, respectively). Now we report that this phenomenon is also a frequent event in laryngeal carcinomas. Of a total of 76 laryngeal carcinomas analyzed, 66% of the tumors showed an alteration in HLA class I phenotype. These altered HLA phenotypes were classified as total HLA loss (10.52%) (phenotype I); HLA-A locus-specific loss (13.15%) (phenotype IIIa); HLA-B locus-specific loss (10.52%) (phenotype IIIb); HLA allelic loss (27.63%) (phenotype IV); and HLA-A and B locus loss (3.9%). Comparison of histopathological parameters with HLA expression showed that poorly differentiated tumors had the lowest levels of HLA class I expression (p < 0.05).

Diversity of HLA-B17 alleles and haplotypes in East Asians and a novel Cw6 allele (Cw*0604) associated with B*5701.

The distribution of HLA-B17 alleles and their association with HLA-A, -C and -DRB1 alleles were investigated in seven East Asian populations Japanese, South Korean, Chinese-Korean, Man, Northern Han, Mongolian and Buryat populations). The B17 alleles were identified from genomic DNA using group-specific polymerase chain reaction (PCR) followed by hybridization with sequence-specific oligonucleotide probes (SSOP). In all of these East Asian populations, except Japanese and Chinese-Koreans, B*5701 was detected and strongly associated with A*0101, Cw*0602 and DRB1*0701. In contrast, B*5801 was detected in all the seven populations and strongly associated with A*3303, Cw*0302, DRB1*0301 and DRB1*1302. The A*3303-Cw*0302-B*5801-DRB1*1302 haplotype was observed in South Korean, Chinese-Korean, Buryat and Japanese populations, while A*3303-Cw*0302-B*5801-DRB1*0301 was predominantly observed in the Mongolian population. A similar haplotype, A*0101-Cw*0302-B*5801-DRB1*1302, was observed in the Buryat population. A novel Cw6 allele, Cw*0604, was identified in the Man population. This Cw allele was observed on the haplotype A*0101-B*5701-DRB1*0701. Thus, we confirmed, at the sequence level, that the common haplotypes carrying B*5701 and B*5801 have been conserved and shared in East Asian populations.

Monomorphic HLA class I-(non-A, non-B) expression on Ewing's tumor cell lines, modulation by TNF-alpha and IFN-gamma.

In this study, the expression of polymorphic and non-polymorphic MHC antigens in Ewing's tumor (ET) cells was examined by surface staining, Western blots and transcriptional analysis. Cell lines derived from Ewing's tumors largely lack polymorphic HLA class Ia antigens of both the HLA-A and the HLA-B loci but binding of monomorphic HLA antibodies indicates significant expression of HLA-C locus antigens and/or HLA class Ib molecules. HLA Ib molecules encoded by the HLA-E, -F or -G loci with a molecular mass of less than 44 kDa were not detected in lysates of either constitutive or TNF-alpha plus IFN-gamma treated ET cells. Two representative ET cell lines with either detectable HLA-A, -B antigens (A673) or absolutely non-detectable HLA-A, -B antigens (SK-ES-1) were further subjected to transcriptional analysis. A673 mRNA hybridized with HLA-A, -B, -C and HLA-E-specific probes in Northern blots. By contrast, mRNA specific for HLA-A, -B, -C was negative in SK-ES-1 but TNF-alpha plus IFN-gamma reconstituted HLA-A, -B, -C transcription in this cell line. HLA-E was transcribed in A673 but not in SK-ES-1. Combining mRNA and surface expression of HLA class Ia molecules results in a highly variable pattern of defective HLA class I expression in this type of neuroectodermal tumor. The involvement of the ET-specific fusion transcript EWS/Fli-1 in modulating the HLA-A and -B locus antigens is likely to occur by the upregulation of c-myc in these tumors. The exceptionally constant expression of HLA-C or some other non-A, non-B antigens (reactive with defined monoclonal antibodies) implies important consequences on tumor-cell resistance against specific CTL and NK activity in vivo.

Kinetics of interaction of HLA-C ligands with natural killer cell inhibitory receptors.

The recognition of HLA-C molecules by specific inhibitory receptors is a crucial step in the regulation of natural killer (NK) cell function. Using soluble, recombinant HLA-C molecules and NK inhibitory receptors (NKIR, members of the immunoglobulin superfamily), we show that HLA-C binds to NKIR molecules with extremely fast association and dissociation rates, among the fastest of the immune system interactions so far studied. These kinetics may be essential for the biological function of NK cells, i.e., to facilitate the rapid immunosurveillance of cells for absent or diminished expression of class I MHC proteins.

A comparison of flow cytometry and immunohistochemistry in human colorectal cancers.

In human colorectal cancer it has been reported that some tumours lack the HLA-ABC antigens. This has been interpreted as reflecting tumour escape from the immune system. Earlier data have been obtained by immunohistochemistry. In this study, we compared the expression of HLA-ABC, HLA-DR, CD80 (B7-1) and CD54 (ICAM-1) in 20 tumours using both a conventional immunohistochemistry two-layer technique and multiparameter flow cytometry, gating on an epithelial cell marker. Colorectal cancer tissue used in flow cytometry was dissociated with collagenase, deoxyribonuclease and hyaluronidase. The intensity of expression of HLA-ABC, HLA-DR and CD80 was unaffected by the enzymes, but CD54 was decreased by 30%. The reproducibility of flow cytometry was good. Microscopy of sections revealed that about 5% of each tumour sample consisted of normal epithelium, but even after correction for this, flow cytometry was superior to immunohistochemistry in 33 out of 80 cases, and showed that tumours described as HLA-ABC negative by immunohistochemistry were in fact weakly positive for HLA-ABC. We conclude that flow cytometry and immunohistochemistry are complementary, and that flow cytometry is superior to immunohistochemistry for detecting antigens/epitopes present in low amounts.

Direct binding of a soluble natural killer cell inhibitory receptor to a soluble human leukocyte antigen-Cw4 class I major histocompatibility complex molecule.

Natural killer (NK) cells expressing specific p58 NK receptors are inhibited from lysing target cells that express human leukocyte antigen (HLA)-C class I major histocompatibility complex molecules. To investigate the interaction between p58 NK receptors and HLA-Cw4, the extracellular domain of the p58 NK receptor specific for HLA-Cw4 was overexpressed in Escherichia coli and refolded from purified inclusion bodies. The refolded NK receptor is a monomer in solution. It interacts specifically with HLA-Cw4, blocking the binding of a p58-Ig fusion protein to HLA-Cw4-expressing cells, but does not block the binding of a p58-Ig fusion protein specific for HLA-Cw3 to HLA-Cw3-expressing cells. The bacterially expressed extracellular domain of HLA-Cw4 heavy chain and beta2-microglobulin were refolded in the presence of a HLA-Cw4-specific peptide. Direct binding between the soluble p58 NK receptor and the soluble HLA-Cw4-peptide complex was observed by native gel electrophoresis. Titration binding assays show that soluble monomeric receptor forms a 1:1 complex with HLA-Cw4, independent of the presence of Zn2+. The formation of complexes between soluble, recombinant molecules indicates that HLA-Cw4 is sufficient for specific ligation by the NK receptor and that neither glycoprotein requires carbohydrate for the interaction.

Unusual uniformity of the N-linked oligosaccharides of HLA-A, -B, and -C glycoproteins.

MHC class I glycoproteins possess an invariant site for N-linked oligosaccharide addition at position 86 of the heavy chain. For human HLA-A, -B, and -C class I glycoproteins, position 86 is the only site of N-linked glycosylation. Comparison of the size and relative abundance of oligosaccharides associated with nine HLA-A, -B, or -C allotypes isolated from EBV-transformed B cell lines and mixtures of HLA-A, -B, and -C allotypes isolated from pooled PBLs revealed a very restricted set of structures. Allotypes encoded by the HLA-A and -B loci have two predominant glycan structures that were almost exclusively di-sialylated. In contrast, HLA-C allotypes have four glycan structures, comprising those associated with HLA-A and -B and two additional glycans. Identical oligosaccharides were present on different allotypes of a class I HLA locus, and in particular, HLA-C allotypes defining two inhibitory specificities for NK cells were shown to possess the same set of oligosaccharides. The uniformity of oligosaccharide structure associated with different HLA-A, -B, and -C products and the relative lack of heterogeneity for any given allotype are unusual features for a mammalian glycoprotein. Particularly striking is that such conserved oligosaccharide structures juxtapose the major regions of amino acid sequence variation within the Ag recognition site, including the polymorphisms of the alpha 1 helix that determine the inhibitory ligands for human NK cells.

Anchored PCR cloning of the novel HLA-Cw*0704 allele detected by PCR-SSP.

The novel HLA-Cw*0704 allele, previously detected as the PCR-SSP variant Cw7/8v, has been cloned and sequenced from the homozygous typing cell KRO3/4 after amplification by anchored PCR. The nucleotide sequence of Cw*0704 is closely related to those of other Cw*07 alleles, but carries specific changes in exon 3 consistent with its serological behavior-a short Cw7 cross-reactive with antibodies directed against HLA-Cw8. Some of the substitutions of Cw*0704 have not been previously described for HLA-C but are found in HLA-B alleles and in published C sequences of non-human primates. The new allele carries a novel polymorphism in its 5' untranslated region (5' ut) that could be shared by all Cw*07 alleles. By PCR-SSP, Cw*0704 is a relatively common allele in English Caucasoids at a frequency of 4.6%. It is most often observed on HLA-B44 haplotypes previously described as HLA-C "blank", although linkage disequilibria with other HLA-B specificities have been found.

Apparent lack of MHC restriction in binding of class I HLA molecules to solid-phase peptides.

The specificity of binding of solubilized, purified HLA-A,B molecules to solid-phase peptides has been examined using the assay described by Bouillet et al. [1989. Nature (Lond.). 339:473.] 64 peptides derived from the sequences of viral antigens, HLA-A,B,C heavy chains, and clathrin light chains were tested for binding to HLA-A2.1, Aw68.1, Aw69, B44, and B5, molecules that differ by 5-17 residues of the peptide binding groove. 15 of the peptides, including those known to be T cell epitopes, gave significant binding. The pattern of peptide binding for each of the five HLA-A,B molecules was not significantly different. Binding was demonstrated to be a property of native beta 2m-associated HLA-A,B molecules that preserved conformational antigenic determinants after binding to peptide. In comparison to our previous results from solution-based assays the proportion of HLA-A,B molecules that can bind solid-phase peptides is very high. This accessibility of solid-phase peptides to the binding site of MHC molecules may be directly related to the observed absence of MHC specificity in the binding.

The structure and expression of genes encoding serologically undetected HLA-C locus antigens.

Approximately 20 to 50% individuals in every race are untypable by human alloantisera for at least one allele of HLA-C locus and the surface expression of HLA-C locus Ag in such an individual (HLA-C blank Ag) remains unknown. To investigate the structure and the surface expression of HLA-C blank Ag, two genes (Cb-1 and Cb-2) encoding HLA-C blank Ag were cloned and their primary structures were determined and compared with other HLA-C locus genes. The similarity of amino acids between Cb-1 and Cw1 was the highest among HLA-C locus genes previously published. Five amino acid substitutions between these molecules were shown to be located on the beta-strand of alpha 1 and alpha 2 domains, suggesting that they might change the conformational allodeterminants on the alpha-helical region of Cw1 which were recognized by antibodies. On the other hand, Cb-2 was the closest to Cw2.2. Six of nine amino acid substitutions between these molecules were observed on alpha 1 and alpha 2 domains, whereas three other substitutions were located on the leader peptide, the alpha 3 domain and the transmembrane. Two substitutions (residues 73 and 163) of the alpha-helical region of the alpha 1 and alpha 2 domains and one (residue 16) of exposed loop may make new allodeterminants which are not recognized by anti-Cw2 sera as well as other alloantisera. The surface expression of these genes was examined on transfected mouse L cells and human B cell line. Both gene products were expressed stably on the surface of these cells. These results suggest that HLA-C blank Ag are most probably expressed on cells in HLA-C blank individuals and that the primary structures of these Ag, which were not detectable by the available alloantisera, may be incapable of generating corresponding alloantibodies.

Sequence and gene transfer analyses of HLA-CwBL18 (HLA-C blank) and HLA-Cw5 genes. Implications for the control of expression and immunogenicity of HLA-C antigens.

Our previous studies suggested that a serologically undetectable HLA-C blank allele (HLA-CwBL18) is either a variant Cw5 allele or a novel HLA-C Ag. To examine these possibilities, the CwBL18 and Cw5 genes from the TCC (HLA-A1, -A2, -B52, -B18, -Cw-, -Cw-) and QBL (HLA-A26, -B18, -Cw5) EBV-transformed B lymphoblastoid cell lines (LCL) were cloned, sequenced, and transferred into HLA-A, -B, -C null LCL mutant .221 cells. The CwBL18 Ag was detected on the cell surface of CwBL18 transferents by flow cytometry with the anti-class I mAb W6/32 but not by complement-mediated cytotoxicity with currently available HLA-C specific antisera. Sequence analysis of the Cw-BL18 gene indicated that the CwBL18 Ag is "C"-like because it contains all C-locus-specific residues and amino acid replacements commonly found in HLA-C alleles. However, the amino acid sequence of the CwBL18 Ag is unusual; CwBL18 lacks unique allele-specific residues when compared with the sequences of other HLA-C alleles. Moreover, apart from the C-locus-specific differences, the sequence of CwBL18 is identical to the HLA class I consensus sequence. This striking homology of CwBL18 to other HLA class I alleles suggests that CwBL18 may be a weak Ag. Taken together, these data demonstrate that CwBL18 is not a variant Cw5 Ag but is a newly described HLA-C Ag. In contrast to CwBL18, the Cw5 Ag is serologically detectable on the cell surface of Cw5 transferents with HLA-specific allo-antisera. Rather unexpectedly, Cw5 was usually expressed at a lower level than CwBL18 on the surface of .221 transferents as evaluated by W6/32 mAb binding analyses. The sequence of Cw5 revealed several unique amino acid replacements. Two of these substitutions, at residue 35 of the alpha 1 domain and residue 275 of the transmembrane domain, may be responsible for the reduced cell surface expression of Cw5. Additional unique replacements at residues 138 and 177 of the alpha 2 domain suggest that these amino acids may be important in the formation of an epitope recognized by a Cw5-specific antibody.

Complete nucleotide sequence of a unique HLA class I C locus product expressed on the human choriocarcinoma cell line BeWo.

We have used Northern blot analysis to detect mRNA from class I HLA genes in the human choriocarcinoma cell line BeWo, which has been previously shown to express an atypical HLA class I molecule, in the absence of HLA A and B. Hybridization was seen with three class I DNA probes, the strongest being seen with the probe pC800, which corresponds to an 800-bp section of the Cw3 gene. We have made cDNA libraries from BeWo cells and screened for positive clones by using class I DNA probes. Of the clones isolated, we determined the complete sequence of one and partial sequence of five shorter clones. They all code for an identical C locus-related product, which does not correspond to published C locus sequences.