Comparison of HLA class I gene sequences. Derivation of locus-specific oligonucleotide probes specific for HLA-A, HLA-B, and HLA-C genes

The major histocompatibility complex in man contains at least 20 class I genes. Included within this family are three closely linked loci with 11-47 codominant alleles that encode the classical transplantation antigens HLA-A, -B, and -C. The study of individual HLA-A, -B, and -C genes is complicated both by the high degree of sequence homology among all members of the class I gene family and by the high degree of polymorphism exhibited by HLA-A, -B, and -C genes. Identification of potential locus-specific regions suitable for use as unique probes has been limited by the small number of nucleotide sequences available for comparison. In the present study, the nucleotide sequences of two cDNA clones, designated HLA-4 and HLA-10, that encode previously unsequenced alleles of HLA-C and HLA-A genes, respectively, are compared with those of other class I genes. From these intergenic and interallelic comparisons, it was deduced that the nucleotide sequence encoding amino acids 291-299 of the transmembrane region showed sufficient divergence between loci and similarity between alleles, to be suitable for the generation of locus-specific probes. Synthetic oligonucleotides were generated and shown to be highly locus-specific in hybridization. These probes were used successfully for the quantitation of the relative amounts of mRNA transcribed in human liver from HLA-A, -B, and -C genes; they should greatly simplify future studies of restriction fragment length polymorphisms of HLA-A, -B, and -C alleles as genetic markers of disease susceptibility.     

Rhesus macaque KIR bind human MHC class I with broad specificity and recognize HLA-C more effectively than HLA-A and HLA-B

Human killer cell immunoglobulin-like receptors (KIR) recognize A3/11, Bw4, C1, and C2 epitopes carried by mutually exclusive subsets of human leukocyte antigen (HLA)-A, -B, and -C allotypes. Chimpanzee and orangutan have counterparts to HLA-A, -B, and -C, and KIR that recognize the A3/11, Bw4, C1, and C2 epitopes, either individually or in combination. Because rhesus macaque has counterparts of HLA-A and -B, but not HLA-C, we expected that rhesus KIR would better recognize HLA-A and -B, than HLA-C. Comparison of the interactions of nine rhesus KIR3D with 95 HLA isoforms, showed the KIR have broad specificity for HLA-A, -B, and -C, but vary in avidity. Considering both the strength and breadth of reaction, HLA-C was the major target for rhesus KIR, followed by HLA-B, then HLA-A. Strong reactions with HLA-A were restricted to the minority of allotypes carrying the Bw4 epitope, whereas strong reactions with HLA-B partitioned between allotypes having and lacking Bw4. Contrasting to HLA-A and -B, every HLA-C allotype bound to the nine rhesus KIR. Sequence comparison of high- and low-binding HLA allotypes revealed the importance of polymorphism in the helix of the α₁ domain and the peptide-binding pockets. At peptide position 9, nonpolar residues favor binding to rhesus KIR, whereas charged residues do not. Contrary to expectation, rhesus KIR bind more effectively to HLA-C, than to HLA-A and -B. This property is consistent with major histocompatibility complex (MHC)-C having evolved in hominids to be a generally superior ligand for KIR than MHC-A and MHC-B.     

Allelic variation in HLA-B and HLA-C sequences and the evolution of the HLA-B alleles

Several new HLA-B (B8, B51, Bw62)- and HLA-C (Cw6, Cw7)-specific genes were isolated either as genomic cosmid or cDNA clones to study the diversity of HLA antigens. The allele specificities were identified by sequence analysis in comparison with published HLA-B and -C sequences, by transfection experiments, and Southern and northern blot analysis using oligonucleotide probes. Comparison of the classical HLA-A, -B, and -C sequences reveals that allele-specific substitutions seem to be rare events. HLA-B51 codes only for one allelespecific residue: arginine at position 81 located on the 1 helix, pointing toward the antigen binding site. HLA-B8 contains an acidic substitution in amino acid position 9 on the first central sheet which might affect antigen binding capacity, perhaps in combination with the rare replacement at position 67 (F) on the ul helix. HLA-B8 shows greatest homology to HLA-Bw42, -Bw41, -B7, and-Bw60 antigens, all of which lack the conserved restriction sites Pst I at position 180 and Sac I at position 131. Both sites associated with amino acid replacements seem to be genetic markers of an evolutionary split of the HLA-B alleles, which is also observed in the leader sequences. HLA-Cw7 shows 98% sequence identity to the JY328 gene. In general, the HLA-C alleles display lower levels of variability in the highly polymorphic regions of the 1 and 2 domains, and have more distinct patterns of locus-specific residues in the transmembrane and cytoplasmic domains. Thus we propose a more recent origin for the HLA-C locus.     

Cloning and analysis of HLA class I cDNA encoding a new HLA-C specificity Cx52

HLA-C loci frequently have an unclassifiable blank (CwBL) specificity. It is unclear whether HLA-C specificities associated with the haplotypes of A24 Bw52 CwBL DR2 DQw1 and Aw33 B44 CwBL DRw13 DQw1 in Japanese (tentatively named Cx52 and Cx44, respectively) really exist. Southern hybridization experiments revealed that restriction enzyme-cleaved genomic DNA from AKIBA, consanguineous HLA homozygote, two other homozygotes with the former haplotype, and three homozygous cells with the latter haplotype hybridized strongly with an HLA-C-specific probe. We have screened the cDNA library constructed from AKIBA to isolate cDNA clones encoding the putative Cx52 antigen, and picked up 103 cDNA clones with HLA-class I DNA probes as possible candidates. By restriction enzyme mapping and Southern hybridization of selected clones, we identified three isotypes of cDNA clones, pA01, pB55, and pC68, which appeared to encode A24, Bw52, and Cx52, respectively. The nucleotide sequence of pC68 showed higher homology with exons of the HLA-C gene than with those of the HLA-A and HLA-B genes, especially in exons 6–8 which include the HLA-C-specific region. Comparison of amino acid sequences showed more than 86% homology among Cw1, Cw2, Cw3, and new pC68-encoded Cx52 proteins. These results support the notion that the inability to define C antigens serologically in this Cx52 haplotype is not due to a HLA-C gene deletion or mutation, but to the absence of typing sera.     

Molecular mapping of a new public HLA class I epitope shared by all HLA-B and HLA-C antigens and defined by a monoclonal antibody

It has previously been shown that a mouse monoclonal antibody, designated 4E, reacts with an epitope common to all HLA-B and -C antigens and those of the HLA-Aw19 cross-reactive group, namely, HLA-A29,-A30, -A31, -A32, -Aw33, and -Aw74. In order to pinpoint the amino acid residues which comprise the public specificity recognized by 4E, an HLA-A29 cDNA clone was isolated and its predicted amino acid sequence compared with those of other clonedHLA class I genes. The isolated HLA-A29 cDNA corresponded to the rarer of the twoA29 variant alleles,A29.1. Two amino acid residues of HLA-A29.1, gln-144 and arg-151, were found in all 24HLA-B andHLA-C alleles examined but were present in only one of 15HLA-A alleles for which sequence data are available. Importantly, this exceptional allele wasHLA-A32, another member of the HLA-Aw19 cross-reactive group. Gln-144 and arg-151 should be capable of jointly contributing to the binding site for 4E, as they are situated in successive alpha-helical subregions and are predicted to be juxtaposed in the three-dimensional HLA molecule. Four other residues in the first or second external domains of HLA-A29.1 (thr-9, leu-62, gln-63, and his-102) were unique among theHLA-A alleles, but none of these was found in corresponding positions ofHLA-B or-C alleles and thus failed to correlate with presence or absence of the 4E determinant. These observations are consistent with the notion that gln-144 and arg-151 define a determinant common to HLA-B, HLA-C, and the HLA-Awl9 cross-reactive group and the binding site of the monoclonal antibody 4E.     

Selective downregulation of rhesus macaque and sooty mangabey major histocompatibility complex class I molecules by Nef alleles of simian immunodeficiency virus and human immunodeficiency virus type 2

Human immunodeficiency virus type 1 (HIV-1) Nef downregulates HLA-A and -B molecules, but not HLA-C or -E molecules, based on amino acid differences in their cytoplasmic domains to simultaneously evade cytotoxic T lymphocyte (CTL) and natural killer cell surveillance. Rhesus macaques and sooty mangabeys express orthologues of HLA-A, -B, and -E, but not HLA-C, and many of these molecules have unique amino acid differences in their cytoplasmic tails. We found that these differences also resulted in differential downregulation by primary simian immunodeficiency virus (SIV) SIV(smm/mac) and HIV-2 Nef alleles. Thus, selective major histocompatibility complex class I downregulation is a conserved mechanism of immune evasion for pathogenic SIV infection of rhesus macaques and nonpathogenic SIV infection of sooty mangabeys.     

DNA Polymorphism of the major histocompatibility class I genes and their association with serologically defined haplotypes

DNA of unrelated persons as well as members of families that were totally or partially homozygous or completely heterozygous on the loci of the major histocompatibility class I genes has been isolated from peripheral blood lymphocytes and blot hybridized with the class I pseudogene pHLA 12.4 probe. The autoradiographic DNA patterns were discussed and compared with well-defined serological features. Positive associations with serologically typed alleles had been demonstrated for HLA-A1,11 ; -A2; -A3; -B7; -B14; -B35;-Bw41; and -Cw5.     

A single bottleneck in HLA-C assembly

Poor assembly of class I major histocompatibility HLA-C heavy chains results in their intracellular accumulation in two forms: free of and associated with their light chain subunit (beta(2)-microglobulin). Both intermediates are retained in the endoplasmic reticulum by promiscuous and HLA-dedicated chaperones and are poorly associated with peptide antigens. In this study, the eight serologically defined HLA-C alleles and the interlocus recombinant HLA-B46 allele (sharing the HLA-C-specific motif KYRV at residues 66-76 of the alpha1-domain alpha-helix) were compared with a large series of HLA-B and HLA-A alleles. Pulse-labeling experiments with HLA-C transfectants and HLA homozygous cell lines demonstrated that KYRV alleles accumulate as free heavy chains because of both poor assembly and post-assembly instability. Reactivity with antibodies to mapped linear epitopes, co-immunoprecipitation experiments, and molecular dynamics simulation studies additionally showed that the KYRV motif confers association to the HLA-dedicated chaperones TAP and tapasin as well as reduced plasticity and unfolding in the peptide-binding groove. Finally, in vitro assembly experiments in cell extracts of the T2 and 721.220 mutant cell lines demonstrated that HLA-Cw1 retains the ability to form a peptide-receptive interface despite a lack of TAP and functional tapasin, respectively. In the context of the available literature, these results indicate that a single locus-specific biosynthetic bottleneck renders HLA-C peptide-selective (rather than peptide-unreceptive) and a preferential natural killer cell ligand.     

Role of the inhibitory KIR ligand HLA-Bw4 and HLA-C expression levels in the recognition of leukemic cells by Natural Killer cells

Transplantation of acute myeloid leukemia (AML) patients with grafts from related haploidentical donors has been shown to result in a potent graft-versus-leukemia effect. This effect is mediated by NK cells because of the lack of activation of inhibitory killer cell immunoglobulin-like receptors (KIRs) which recognize HLA-Bw4 and HLA-C alleles. However, conflicting results have been reported about the impact of KIR ligand mismatching on the outcome of unrelated HLA-mismatched hematopoietic stem cells transplants (HSCT) to leukemic patients. The interpretation of these conflicting results is hampered by the scant information about the level of expression of HLA class I alleles on leukemic cells, although this variable may affect the activation of inhibitory KIRs. Therefore in the present study, utilizing a large panel of human monoclonal antibodies we have measured the level of expression of HLA-A, -B and -C alleles on 20 B-chronic lymphoid leukemic (B-CLL) cell preparations, on 16 B-acute lymphoid leukemic (B-ALL) cell preparations and on 19 AML cell preparations. Comparison of the level of HLA class I antigen expression on leukemic cells and autologous normal T cells identified selective downregulation of HLA-A and HLA-B alleles on 15 and 14 of the 20 B-CLL, on 2 and 5 of the 16 B-ALL and on 7 and 11 of the 19 AML patients tested, respectively. Most interestingly HLA-C alleles were markedly downregulated on all three types of leukemic cells; the downregulation was most pronounced on AML cells. The potential functional relevance of these abnormalities is suggested by the dose-dependent enhancement of NK cell activation caused by coating the HLA-HLA-Bw4 epitope with monoclonal antibodies on leukemic cells which express NK cell activating ligands. Our results suggest that besides the HLA and KIR genotype, expression levels of KIR ligands on leukemic cells should be included among the criteria used to select the donor-recipient combinations for HSCT.     

Structural relatedness of distinct determinants recognized by monoclonal antibody TP25.99 on beta 2-microglobulin-associated and beta 2-microglobulin-free HLA class I heavy chains

The association of HLA class I heavy chains with beta2-microglobulin (beta2m) changes their antigenic profile. As a result, Abs react with either beta2m-free or beta2m-associated HLA class I heavy chains. An exception to this rule is the mAb TP25.99, which reacts with both beta2m-associated and beta2m-free HLA class I heavy chains. The reactivity with beta2m-associated HLA class I heavy chains is mediated by a conformational determinant expressed on all HLA-A, -B, and -C Ags. This determinant has been mapped to amino acid residues 194-198 in the alpha3 domain. The reactivity with beta2m-free HLA class I heavy chains is mediated by a linear determinant expressed on all HLA-B Ags except the HLA-B73 allospecificity and on <50% of HLA-A allospecificities. The latter determinant has been mapped to amino acid residues 239-242, 245, and 246 in the alpha3 domain. The conformational and the linear determinants share several structural features, but have no homology in their amino acid sequence. mAb TP25.99 represents the first example of a mAb recognizing two distinct and spatially distant determinants on a protein. The structural homology of a linear and a conformational determinant on an antigenic entity provides a molecular mechanism for the sharing of specificity by B and TCRs.     

HLA class I polymorphism in the Albanian population

The HLA class I polymorphism was studied in a sample of the Albanian population. Ninety-three unrelated healthy Albanians were typed for HLA-A, -B and -Cw antigens by standard microlyphocytotoxicity test. The antigens with the highest frequencies were: HLA-A2 (34.4%), A3 (14.5%) and A1 (12.4%); B51 (19.3%), B35 (12.4%) and B18 (10.2%); Cw4 (16.2%), Cw7 (16.2%) and Cw6 (10.8%). The HLA haplotypes with high frequency in Albanians included A2-B51 (4.3%), A2-B18 (2.4%), A2-B35 (2.4%), Cw4-B35 (7.6%), and Cw7-B18 (6.5%), which are not significantly different from the other neighboring populations. Low frequency of HLA-A1-B8 haplotype (1.1%) is noted in the Albanian population. The frequency of HLA-B27 antigen (1.1%) is one of the lowest frequencies observed in Caucasians. Such results are important in studies of HLA-A1-B8, HLA-B27 and disease associations. These findings should be also useful in understanding the origin of Albanians, representing a base for future studies about HLA polymorphism in the Albanian population.     

Reappraisal of the relationship between the HIV-1-protective single-nucleotide polymorphism 35 kilobases upstream of the HLA-C gene and surface HLA-C expression

Previous studies have found an association between a single-nucleotide polymorphism 35 kb upstream of the HLA-C locus (-35 SNP), HLA-C expression, and HIV-1 set point viral loads. We show that the difference in HLA-C expression across -35 SNP genotypes can be attributed primarily to the very low expression of a single allelic product, HLA-Cw7, which is a common HLA type. We suggest that association of the -35 SNP and HIV-1 load manifests as a result of linkage disequilibrium of this polymorphism with both favorable and unfavorable HLA-C and -B alleles.     

Correlation between HLA antigens with clinical features of mixed infection of hepatitis A and HBV-carriers

It is showed that HAV+HBV mixed infection is a genetically determined pathology. Following HLA-antigens were immunogenetic markers of the disease: HLA-A10, B21, Cw2, Cw5, A10-A19, 88-813, B21-B35, A3-821, A9-B21. Lower risk of disease development was associated with HLA-B5, A2-Cw3, A3-Cw4, B35-Cw4, A3-B35-Cw4. Atypical forms of the hepatitis A were often met in carriers of HLA-Cw5, 827-835, A3-814, A3-B21, A9-B21, whereas typical forms - in carriers of HLA-A10, Cw2, A10-A19, B8-813, 821-835. Mild forms of hepatitis A were associated with the presence of HLA-A10,B22, A10-A19, B8-B13, A3-B21, A9-B8, A10-814, A10-822, A10-Cw3 in the patients' phenotype, whereas intermediate and severe forms - with the presence of HLA-B17, 817-818, 821 - 835, A28-B21, B18-Cw2. The findings about distribution of HLA-antigens and their combinations in mixed hepatitis A+B can be used in attempt of their prediction and prevention.     

"Silent" alleles at the HLA-C locus.

Defined specificities of the HLA-C locus possess a significantly lower immunogenicity as compared with the HLA-A and HLA-B specificities. HLA-C "blanks" are not immunogenic at all. Among the possible explanations, the most likely one appears to be a low phenotypic expression of these alleles. C locus antigens as a whole are thus likely to be of minor relevance in donor/recipient matching for transplantation.     

HSV-2 Specifically Down Regulates HLA-C Expression to Render HSV-2-Infected DCs Susceptible to NK Cell Killing

Both NK cells and CTLs kill virus-infected and tumor cells. However, the ways by which these killer cells recognize the infected or the tumorigenic cells are different, in fact almost opposite. CTLs are activated through the interaction of the TCR with MHC class I proteins. In contrast, NK cells are inhibited by MHC class I molecules. The inhibitory NK receptors recognize mainly MHC class I proteins and in this regard practically all of the HLA-C proteins are recognized by inhibitory NK cell receptors, while only certain HLA-A and HLA-B proteins interact with these receptors. Sophisticated viruses developed mechanisms to avoid the attack of both NK cells and CTLs through, for example, down regulation of HLA-A and HLA-B molecules to avoid CTL recognition, leaving HLA-C proteins on the cell surface to inhibit NK cell response. Here we provide the first example of a virus that through specific down regulation of HLA-C, harness the NK cells for its own benefit. We initially demonstrated that none of the tested HSV-2 derived microRNAs affect NK cell activity. Then we show that surprisingly upon HSV-2 infection, HLA-C proteins are specifically down regulated, rendering the infected cells susceptible to NK cell attack. We identified a motif in the tail of HLA-C that is responsible for the HSV-2-meduiated HLA-C down regulation and we show that the HLA-C down regulation is mediated by the viral protein ICP47. Finally we show that HLA-C proteins are down regulated from the surface of HSV-2 infected dendritic cells (DCs) and that this leads to the killing of DC by NK cells. Thus, we propose that HSV-2 had developed this unique and surprising NK cell-mediated killing strategy of infected DC to prevent the activation of the adaptive immunity.