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The HLA-B15 typing by serological approaches defined the serological subgroups (or splits) B62, B63, B75, B76, B77 and B70 (B71 and B72). The scarcity of sera with specific anti-HLA antibodies makes the serological typing method difficult to discriminate a high variety of HLA antigens, especially between the B15 antigen subgroups. Advancements in DNA-based technologies have led to a switch from serological typing to high-resolution DNA typing methods. DNA sequencing techniques assign B15 specificity to all alleles in the HLA-B*15 allele group, without distinction of the serological split equivalents. However, the presence of antibodies in the patient defined as split B15 antigens urges the identification of HLA-B*15 allele subtypes of the donor, since the presence of donor-specific antibodies is an important contraindication for organ transplantation. Although the HLA dictionary comprises information regarding the serological subtypes of HLA alleles, there are currently 394 B15 antigens out of 516 in the IPD-IMGT/HLA database (3.38.0) without any assigned serological subtype. In this regard, we aimed to identify specific amino acid patterns for each B*15 serological split, in order to facilitate the assignment of B*15 alleles to serological equivalents after high-resolution molecular typing. As a result, serological specificities of 372/394 not yet assigned alleles could be predicted based on amino acid motifs. Furthermore, two new serological types were identified and added, B62-Bw4 and B71-Bw4. Electronic supplementary material The online version of this article (10.1007/s00251-020-01172-8) contains supplementary material, which is available to authorized users.

Peptide selection in infected cells is not fully understood yet, but several indications point to the fact that there are differences to uninfected cells, especially in productive HCMV infection, since HCMV evolved various strategies to disable the hosts immune system, including presentation of peptide-HLA complexes to immune effector cells. Therefore, peptide predictions for specific HLA alleles are limited in these cases and the naturally presented peptide repertoire of HCMV-infected cells is of major interest to optimize adoptive T cell therapies. The allotypes HLA-B*35:01 and B*35:08 differ at a single amino acid at position 156 and have been described to differ in their peptide features and in their association with the peptide loading complex. Virus specific T cells recognizing the allelic pHLA-B*35 complexes could be detected, indicating a significant role of this HLA subtypes in viral immunity. However, naturally selected and presented viral peptides have not been described so far. In this study, we analyzed the peptide binding repertoire for HLA-B*35:01 and HLA-B*35:08 in HCMV-infected cells. The isolated peptides from both allelic subtypes were of extraordinary length, however differed in their features, origin, and sequence. For these HCMV-originated peptides, no overlap in the peptide repertoire could be observed between the two allelic subtypes. These findings reveal the discrepancies between predicted and naturally presented immunogenic epitopes and support the need of comprehensive peptide recruitment data for personalized and effective cellular therapies.

Defining permissive and non-permissive mismatches for transplantation is a demanding challenge. Single mismatches at amino acid (AA) position 156 of human leucocyte antigen (HLA) class I have been described to alter the peptide motif, repertoire, or mode of peptide loading through differential interaction with the peptide-loading complex. Hence, a single mismatch can tip the balance and trigger an immunological reaction. HLA-B*35 subtypes have been described to evade the loading complex, 156 mismatch distinguishing B*35:01 and B*35:08 changes the binding groove sufficiently to alter the sequence features of the selected peptide repertoire. To understand the functional influences of residue 156 in B*35 variants, we analyzed the peptide binding profiles of HLA-B*35:01156Leu, B*35:08156Arg and B*35:62156Trp. The glycoprotein tapasin represents a target for immune evasions and functions within the multimeric peptide-loading complex to stabilize empty class I molecules and promote acquisition of high-affinity peptides. All three B*35 subtypes showed a tapasin-independent mode of peptide acquisition. HLA-B*35-restricted peptides of low- and high-binding affinities were recovered in the presence and absence of tapasin and subsequently sequenced utilizing mass spectrometry. The peptides derived from B*35 variants differ substantially in their features dependent on their mode of recruitment; all peptides were preferentially anchored by Pro at p2 and Tyr, Phe, Leu, or Lys at pΩ. However, the Trp at residue 156 altered the p2 motif to an Ala and restricted the pΩ to a Trp. Our results highlight the importance of understanding the impact of key micropolymorphism and how a single AA mismatch orchestrates the neighboring AAs. Electronic supplementary material The online version of this article (doi:10.1007/s00251-015-0896-4) contains supplementary material, which is available to authorized users.

Supertypes are groups of human leukocyte antigen (HLA) alleles which bind overlapping sets of peptides due to sharing specific residues at the anchor positions—the B and F pockets—of the peptide-binding region (PBR). HLA alleles within the same supertype are expected to be functionally similar, while those from different supertypes are expected to be functionally distinct, presenting different sets of peptides. In this study, we applied the supertype classification to the HLA-A and HLA-B data of 55 worldwide populations in order to investigate the effect of natural selection on supertype rather than allelic variation at these loci. We compared the nucleotide diversity of the B and F pockets with that of the other PBR regions through a resampling procedure and compared the patterns of within-population heterozygosity (He) and between-population differentiation (GST) observed when using the supertype definition to those estimated when using randomized groups of alleles. At HLA-A, low levels of variation are observed at B and F pockets and randomized He and GST do not differ from the observed data. By contrast, HLA-B concentrates most of the differences between supertypes, the B pocket showing a particularly high level of variation. Moreover, at HLA-B, the reassignment of alleles into random groups does not reproduce the patterns of population differentiation observed with supertypes. We thus conclude that differently from HLA-A, for which supertype and allelic variation show similar patterns of nucleotide diversity within and between populations, HLA-B has likely evolved through specific adaptations of its B pocket to local pathogens. Electronic supplementary material The online version of this article (doi:10.1007/s00251-015-0875-9) contains supplementary material, which is available to authorized users.

HLA class I molecules and killer cell immunoglobulin-like receptors (KIR) form a diverse system of ligands and receptors that individualize human immune systems in ways that improve the survival of individuals and populations. Human settlement of Oceania by island-hopping East and Southeast Asian migrants started ~3,500 years ago. Subsequently, New Zealand was reached ~750 years ago by ancestral Māori. To examine how this history impacted KIR and HLA diversity, and their functional interaction, we defined at high resolution the allelic and haplotype diversity of the 13 expressed KIR genes in 49 Māori and 34 Polynesians. Eighty KIR variants, including four ‘new’ alleles, were defined, as were 35 centromeric and 22 telomeric KIR region haplotypes, which combine to give >50 full-length KIR haplotypes. Two new and divergent variant KIR form part of a telomeric KIR haplotype, which appears derived from Papua New Guinea and was probably obtained by the Asian migrants en route to Polynesia. Māori and Polynesian KIR are very similar, but differ significantly from African, European, Japanese, and Amerindian KIR. Māori and Polynesians have high KIR haplotype diversity with corresponding allotype diversity being maintained throughout the KIR locus. Within the population, each individual has a unique combination of HLA class I and KIR. Characterizing Māori and Polynesians is a paucity of HLA-B allotypes recognized by KIR. Compensating for this deficiency are high frequencies (>50 %) of HLA-A allotypes recognized by KIR. These HLA-A allotypes are ones that modern humans likely acquired from archaic humans at a much earlier time.

There have been several attempts over the years to identify positions in the peptide-binding region (PBR) of human leukocyte antigens (HLA) that influence the specificity of bound amino acids (AAs) at each position in the peptide. Originally, six pockets (A-F) were defined by calculating the surface area of the PBR on the crystal structure of HLA-A2 molecules. More recent crystallographic analyses of a variety of HLA alleles have led to broader pocket definitions. In this study, we examined the peptide-binding specificity of HLA-B*41 alleles and compared our results with the available pocket definitions. By generating recombinant HLA-B molecules and studying the eluted peptides by mass spectrometry and pool sequencing, we detected two different POmega peptide motifs within the B*41 group: Leu vs Val/Pro. Specificity was dependent on the presence of Leu (B*4102, B*4103, and B*4104) vs Trp (B*4101, B*4105, and B*4106) at AA position 95 in the HLA molecule, whose impact on POmega has been a subject of controversy in current pocket definitions. In contrast, the Arg97Ser mutation did not affect pocket F binding specificity in B*41 subtypes although residue 97 was previously identified as a modulator of peptide binding for several HLA class I alleles. According to most pocket definitions, this study shows that the Asn80Lys substitution in B*4105 impels the peptide's POmega anchor toward more promiscuity. Our sequencing results of peptides eluted from HLA-B*41 variants demonstrate the limitations of current pocket definitions and underline the need for an extended peptide motif database for improved understanding of peptide-major histocompatibility complex interactions.

Analysis of peptides derived from HLA class I molecules indicates that thousands of unique peptides are bound by a single molecular type, and sequence examination of the pooled constituents yields a motif which collectively defines the peptides bound by a given class I molecule. Motifs resulting from pooled sequencing are then used to infer whether particular viral and tumor protein fragments might serve as class I-presented peptide therapeutics. Still undetermined from a pooled motif is the breadth or range of peptides in the population which are brought together to form the pooled motif, and it is therefore not yet known how representative of the population a pooled motif is. By employing hollow fiber bioreactors for large-scale production of HLA class I molecules, sufficient peptides are produced to investigate individual subsets of peptides comprising a motif. Edman sequencing and mass spectrometric analysis of peptides eluted from HLA-B*1501 reveal that many peptide sequences fail to align with either the N- or C-terminal anchors predicted for the B*1501 peptide motif through whole pool sequencing. These analyses further reveal auxiliary anchors not previously detected and peptides significantly larger and smaller than the predicted nonamer, ranging from 6 to 12 amino acids in length. These results demonstrate that constituents of the B*1501 peptide pool vary markedly in comparison with one another and therefore in comparison with previously established B*1501 motifs, and such complexity indicates that many of the peptide ligands presented to CTL cannot be predicted using class I consensus motifs as search criteria.

The identification of expression variants is a challenge in HLA diagnostics. We here describe the identification of the novel allele HLA-B*3565Q. The serological HLA class I type, as determined by a lymphocytotoxicity test, was A11,24; B38; Bw4; Cw-; whereas PCR-sequence-specific primers resulted in A*11,*24, B*35,*38; Cw*12, thus suggesting the presence of a nonexpressed B*35 allele. To clarify the lack of serological HLA-B35 reactivity, exons 2 and 3 were sequenced following haplotype-specific amplification. At position 564 from the beginning of the coding region (exon 3), a transversion (C-->G) was observed, which, at the amino acid level, results in a substitution from cysteine to tryptophane at position 164 of the mature polypeptide. Because this position is essential for the formation of a disulfide bond linking the cysteine residues at positions 101 and 164, which is strongly conserved in functional class I molecules of vertebrates, the disruption of this bond is very likely to be the reason for the lack of serological detectability. We later found the same novel allele in a second unrelated individual, of whom we were able to establish a lymphoblastoid cell line (B-LCL). Serological testing of this B-LCL indicated a very low aberrant expression of HLA-B*3565Q, which cannot be expected to be detected by standard serology techniques.

DNA variants in the tumor necrosis factor-α (TNF) and linked lymphotoxin-α genes, and specific alleles of the highly polymorphic human leukocyte antigen B (HLA-B) gene have been implicated in a plethora of immune and infectious diseases. However, the tight linkage disequilibrium characterizing the central region of the human major histocompatibility complex (MHC) containing these gene loci has made difficult the unequivocal interpretation of genetic association data. To alleviate these difficulties and facilitate the design of more focused follow-up studies, we investigated the structure and distribution of HLA-B-specific MHC haplotypes reconstructed in a European population from unphased genotypes at a set of 25 single nucleotide polymorphism sites spanning a 66-kilobase long region across TNF. Consistent with the published data, we found limited genetic diversity across the so-called TNF block, with the emergence of seven common MHC haplotypes, termed TNF block super-haplotypes. We also found that the ancestral haplotype 8.1 shares a TNF block haplotype with HLA-B*4402. HLA-B*5701, a known protective allele in HIV-1 pathogenesis, occurred in a unique TNF block haplotype.

The high level of polymorphism in major histocompatibility complex (MHC) molecules leads to many allele-specific peptide binding repertoires that can generally be characterized by sequence motifs. Such motifs have previously been elucidated experimentally for several MHC molecules and shown to bind in specificity pockets in the antigen binding cleft. Here, a new and less restrictive description of the traditional antigen binding pockets is derived. These regions are referred to as peptide binding environments and are defined as those residues in a fixed neighborhood of the peptide residues in known crystal structure complexes. By examining the antigen binding environments from MHC molecules with known motifs, we made predictions as to likely motifs for other MHC molecules which share the same environments. The predictions are presented in the form of Tables and are pertinent to class I HLA-A, HLA-B, and HLA-C MHC sequences, and are shown to correlate well with experiments.

The polymorphism of major histocompatibility complex class I chain-related gene B (MICB) and variations in MICB alleles in a variety of populations have been characterized using several genotyping approaches. In the present study, a novel polymerase chain reaction sequence-based typing (PCR-SBT) method was established for the genotyping of MICB exons 2-6, and the allelic frequency of MICB in the Zhejiang Han population was investigated. Among 400 unrelated healthy Han individuals from Zhejiang Province, China, a total of 20 MICB alleles were identified, of which MICB*005:02:01, MICB*002:01:01, and MICB*004:01:01 were the most predominant alleles, with frequencies of 0.57375, 0.1225, and 0.08375, respectively. Nine MICB alleles were detected on only one occasion, giving a frequency of 0.00125. Of the 118 distinct MICB ∼ HLA-B haplotypes identified, 42 showed significant linkage disequilibrium (P 0.05). Haplotypes MICB*005:02:01 ∼ B*46:01, MICB*005:02:01 ∼ B*40:01, and MICB*008 ∼ B*58:01 were the most common haplotypes, with frequencies of 0.0978, 0.0761, and 0.0616, respectively. Five novel alleles, MICB*005:07, MICB*005:08, MICB*027, MICB*028, and MICB*029 were identified. Compared with the MICB*005:02:01 sequence, a GA substitution was observed at nucleotide position 210 in MICB*005:07, and a 1,134 TC substitution in MICB*005:08 and an 862 GA substitution in MICB*027 were detected. In addition, it appears that MICB*028 probably arose from MICB*004:01:01 with an A to G substitution at position 1,147 in exon 6. MICB*029 had a GT transversion at nucleotide position 730 in exon 4, compared with that of MICB*002:01:01. On the basis of the new PCR-SBT assay, these observed results demonstrated MICB allelic variations in the Zhejiang Han population.

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 α(1) 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.

The polymorphism of major histocompatibility complex (MHC) class I HLA-A, -B, and -C molecules may have evolved through pathogen-driven selection of alleles with diverse peptide-binding specificities. Two MHC-encoded molecules that are distantly related to class I, MIC-A and MIC-B, do not function in the presentation of pathogen-derived peptides to T cells with alphabeta T-cell receptors (TCRs), but are broadly recognized by intraepithelial T cells with gammadelta TCRs. However, both MIC-A and MIC-B are polymorphic, displaying an unusual distribution of a number of variant amino acids in their extracellular alpha1, alpha2, and alpha3 domains. In order to further define the polymorphism of MIC-A, we examined its alleles among 275 individuals with common and rare HLA genotypes. Of 16 previously defined alleles, 12 were confirmed and 5 new alleles were identified. A two-by-two analysis of MIC-A and HLA-B alleles uncovered a number of statistically significant associations. These results confirm and extend previous knowledge on the polymorphism of MIC-A. The strong positive linkage of certain MIC-A and HLA-B alleles may have implications for studies related to MHC-associated diseases and transplantation.

HLA-B*0801 is unique among HLA-B allotypes in having dominant amino acid anchors at positions 3 and 5 of the peptide-binding motif. HLA-B*0802 is a variant of HLA-B*0801 in which the Bw6 sequence motif is replaced by a Bw4 sequence motif. This change, involving substitutions at positions 77, 80, 81, 82, and 83 of the B*08 heavy chain, is probably the result of a single evolutionary event of interallelic conversion. Moreover, the difference between B*0802 and B*0801 is sufficient to stimulate a cytotoxic T-cell response. To assess further the functional impact of the Bw4 motif on a B8 background, we compared the peptide-binding specificity of the B*0801 and B*0802 allotypes by sequencing the mixture of peptides endogenously bound to B*0802 and 12 individual peptides purified from that mixture. The HLA-B*0802 allotype, while able to bind some peptides bound by B*0801, has a broader repertoire of endogenously bound peptides than B*0801: the peptides bound by B*0802 are more variable in length and exhibit greater diversity in the carboxyl-terminal amino acid which interacts with the F pocket.

The binding of 303 8-mer to 11-mer peptides carrying the anchor residues at P2 and the C-terminus to HLA-B*5101 molecules was examined by a stabilization assay in which peptides were incubated with RMA-S-B*5101 cells at 26 degrees C for 3 h. Analysis of the binding of these peptides to HLA-B*5101 molecules showed that Pro and Ala at P2, and Ile, Val, and Leu at the C-terminus functioned as anchor residues, while Gly at P2 and Met at the C-terminus were weak anchors. Pro was a stronger anchor residue than Ala at P2, while Ile was the strongest anchor at the C-terminus. Among 8-mer to 11-mer peptides, the 9-mer peptides showed the strongest binding to HLA-B*5101 molecules. This is in contrast to our recent findings that 10-mer and 11-mer peptides bind to HLA-B*3501 molecules as effectively as 9-mer peptides. Since both HLA class I molecules have the same B-pocket and the binding peptides carry the same anchor residues, it is assumed that the structure of the F-pocket may restrict the length of binding peptides. The ability of HLA-B*5101 binding peptides to stabilize the HLA-B*5101 molecules was markedly lower than that of HLA-B*3501 binding peptides to stabilize the HLA-B*3501 molecules. It is known that HLA-B*5101 is a slow assembling molecule, while HLA-B*3501 assembles rapidly. The results imply that the slow assembling of HLA-B*5101 molecules results from the low affinity of peptides to HLA-B*5101 molecules.

HLA-B*3501 is associated with subacute thyroiditis and fast progression of AIDS. An important prerequisite to investigate the T-cell recognition of HLA-B*3501-restricted antigens is the characterization of peptide-HLA-B*3501 interactions. In this study, peptide-HLA-B*3501 interactions were determined in quantitative peptide binding assays. The results were statistically analyzed to evaluate the influence of both anchor and nonanchor positions and the predictability of peptide binding. The binding data demonstrated that all anchor residues at position 2 and the C-terminus found in 9-mers functioned equally as anchors in 10-mers and 11-mers. These minimum requirements of peptide binding were refined by assessing positive and negative effects of nonanchor residues. Aliphatic hydrophobic residues at positions 3, 5, and 8 of 10-mers and position 3 of 11-mers significantly enhanced HLA-B*3501 binding. Similar effects rendered aromatic, bulky residues, acidic or polar residues of 11-mers at position 1 as well as at positions 4, 8, and 10, respectively. Negative effects were observed for residues carrying positively charged side-chains at position 7 of 11-mers. The refined HLA-B*3501 peptide binding motifs enhanced the identification of potential T-cell epitopes. The disparity between positive effects at the middle and C-terminal part (positions 5 – 8 and 10) of 11-mers and shorter peptides supports the extrusion of 11-mer residues at positions 5, 6, and 7, away from the HLA-B*3501 binding cleft.

Routine serological HLA typing of s Syrian family revealed a Bw4-associated HLA-B blank antigen showing Mendelian segregation together with the haplotype A1, Cw2, DR11, DQ7 (a father and one of his children, cells NO. 5958641 and No. 1958125). A more extensive serological analysis was done by using additional polyclonal and monoclonal antibodies (mAb; One-Lambda BL-60 and LM172 plates) as well as the 12th International Workshop class I mAb plate. Both cells showed no conclusive typing reactions with sera towards HLA-B27 and HLA-B37 antigens. Two mAb, PAMELA (27,44,38) and FAY (13,27,37,47), were able to recognize this antigen. The great majority of polyclonal reagents against B37 showed negative reactions, while weak results were frequently observed with anti-B27 allosera. 8 refs., 1 fig., 1 tab.

Studies of the major histocompatibility complex (MHC) in mouse indicate that the recombination sites are not randomly distributed and their occurrence is haplotype-dependent. No data concerning haplotype-specific recombination sites in human are available due to the low number of informative families. To investigate haplotype-specific recombination sites in human MHC, we here describe an approach based on identification of recombinant haplotypes derived from one conserved haplotype at the population level. The recombination sites were mapped by comparing polymorphic markers between the recombinant and assumed original haplotypes. We tested this approach on the extended haplotype HLA A3; B47; Bf*F; C4A*1; C4B*Q0; DR7, which is most suitable for this analysis. First, it carries a number of rare markers, and second, the haplotype, albeit rare in the general population, is frequent in patients with 21-hydroxylase (21OH) defect. We observed recombinants derived from this haplotype in patients with 21OH defect. All these haplotypes had the centromeric part (from Bf to DR) identical to the original haplotype, but they differed in HLA A and B. We therefore assumed that they underwent recombinations in the segment that separates the Bf and HLA B genes. Polymorphic markers indicated that all break points mapped to two segments near the TNF locus. This approach makes possible the mapping of preferential recombination sites in different haplotypes.

The HLA haplotype B18-DR3 has a widespread geographical distribution, but has its greatest frequencies in Southern Europe, probably vestigial of the earliest populations of this region, particularly in the Pays Basque and Sardinia. This haplotype is of medical significance, being that most implicated as a factor of risk in insulin-dependent diabetes mellitus. In this study, the closely linked microsatellite markers (TNFa,b,c) in the region of the tumor necrosis factor (TNF) genes have been used in an attempt to subtype this haplotype in the two populations and/or in healthy and diabetic populations. A total of 79 HLA-B18-DR3 haplotypes were analyzed: 54 in Basques (12 from healthy individuals and 42 from diabetics or their first-degree relatives) and 25 in Sardinians (13 from healthy and 12 from diabetic individuals). The TNF haplotype a1-b5-c2 is completely associated with B18-DR3 in both populations. The homogeneity of the B18-DR3 haplotype in two ethnically pure populations implies stability in evolution, which suggests that the mutation rate of these microsatellite markers must be less than is usually assumed (i. e., approximately 5 x 10(-4) per site per generation). Such markers should be powerful tools for studying genetic drift and admixture of populations, but it remains to be established whether this stability is a rule for all microsatellites in HLA haplotypes or whether it is restricted to some microsatellites and/or some HLA haplotypes. The population genetics of those microsatellites associated with HLA B18-DR3 was also studied in a random sample of the Basque population.

Recent data suggest that HLA-B locus alleles can evolve quickly in native South American populations. To investigate further this phenomenon of new HLA-B variants among Amerindians, we studied samples from another South American tribe, the Cayapa from Ecuador. We selected individuals for HLA-B molecular typing based upon their HLA class II typing results. Three new variants of HLA-B39 and one new variant of HLA-B15 were found in the Cayapa: HLA-B*3905, HLA-B*3906, HLA-B*3907, and HLA-B*1522. A total of thirteen new HLA-B alleles have now been found in the four South American tribes studied. Each of these four tribes studied, including the Cayapa, had novel alleles that were not found in any of the other tribes, suggesting that many of these new HLA-B alleles may have evolved since the Paleo-Indians originally populated South America. Each of these 13 new alleles contained predicted amino acid replacements that were located in the peptide binding site. These amino acid replacements may affect the sequence motif of the bound peptides, suggesting that these new alleles have been maintained by selection. New allelic variants have been found for all common HLA-B locus antigenic groups present in South American tribes with the exception of B48. In spite of its high frequency in South American tribes, no evidence for variants of B48 has been found in all the Amerindians studied, suggesting that B48 may have unique characteristics among the B locus alleles.

Genetic and physical data relating to the human major histocompatibility complex (MHC) were compiled and analyzed, using a genome analysis program. The current contents of the database include: 1) location of over 100 genes and other markers; 2) location of over 250 YAC and cosmid clones; 3) 150 kilobases of genomic DNA sequence including full annotation (exon/intron boundaries, repeats, promoters, etc.); 4) cDNA sequences of currently-known class I and class II alleles; and 5) accompanying descriptive data — references, comments, laboratory addresses, and so on. The database allows rapid access, retrieval, and display of all these data, which should make it a useful tool for the study of the human MHC. MHCDB is publicly available and will be updated as new data become available.

HLA-B73, first described by Mayr and Kirnbauer (1981), is a poorly characterized allospecificity, serologically related to the B7-CREG. We polymerase chain reaction-amplified, cloned and sequenced the HLA-B alleles of the B-LCL LE023, established from a Spanish Caucasoid individual expressing HLA-B73. 5 refs., 2 figs.

The association of HLA A1, B8, and DR3 with generalized myasthenia gravis (GMG) in Caucasoids is well established, but no particular gene has been implicated and there is still no adequate explanation in functional terms. In this study we have taken advantage of sequential genomic markers between B8 and DR3 so as to map the location of susceptibility gene(s) on the A1, B8, DR3 (8.1) ancestral haplotype. By studying 51 patients, we have delineated a region between HLA B and TNF which is shared by 29/29 patients with B8 and DR3, 19/19 patients with B8 but not DR3 and 2/3 patients with DR3 but not B8. The potential importance of this region was confirmed by examining a similar disease induced by D-Penicillamine (D-PenMG) and associated with different HLA class II alleles (DR1 and/or DR7). Among these patients, 7/16 (44%) have B8, often with other markers of 8.1. These results implicate at least two categories of genes in determining susceptibility to MG; one located in the region between HLA B and TNF may be immunoregulatory, whereas the second, located in the class II region, may relate to the inducing factor (e.g., DR1 or DR7 in D-PenMG).

Genomic probes from the HLA-B region of the major histocompatibility complex (MHC) were used to study the association of restriction fragment length polymorphisms (RFLPs) with various MHC alleles, complotypes, and extended haplotypes. The two DNA probes, M20A and R5A, were derived from previously cloned cosmids and are located 38 and 110 kilobases (kb) centromeric to HLA-B, respectively. Five different RFLP variants occurring in five different haplotypic combinations were detected within a panel of 40 homozygous-typing cells and cells from 21 families using Bst EII. In two informative families with HLA-B/DR recombinations the inheritance of the RFLP variants was consistent with their mapping between HLA-B and complotypes. The R5A/M20A haplotypic pattern 6.5 kb/3.0 kb (A) had a normal Caucasian frequency of approximately 0.43 and was found in all independent examples of the extended haplotypes [HLA-B8,SC01,DR3], [HLA-B18,F1C30,DR3], [HLA-Bw62,SC33,DR4], [HLA-B44,SC30,DR4], and [HLA-Bw47,FC91,0,DR7]. The patterns 6.9 kb/3.0 kb (B), 6.5 kb/4.7 kb (C), 1.45 kb/3.0 kb (D), and 6.9 kb/4.7 kb (E) had normal Caucasian frequencies of approximately 0.23, 0.15, 0.15, and 0.04 and were found on all independent examples of [HLA-B38,SC21,DR4], [HLA-Bw57,SC61,DR7], [HLA-B7,SC31,DR2], and [HLA-B44,FC31,DR7], respectively. Individual complotypes or HLA-B alleles which were markers of extended haplotypes showed variable associations. For example, HLA-B7 and the complotype SC31 were associated with all R5A/M20A RFLP haplotypes except haplotype E, although [HLA-B7,SC31,DR7] was associated exclusively with haplotype D. HLA-B27, not known to be part of an extended haplotype, was surprisingly exclusively associated with the 6.5 kb/4.7 kb or C haplotypic pattern in all five instances tested. These findings support the concept of regional conservation of DNA on independent examples of extended haplotypes. The results also further characterize these haplotypes.

We describe the finding of an Alu repeat dimorphism within the first intron of the MICB gene. The frequencies of the two AluyMICB alleles, AluyMICB*0(absence of insertion) and AluyMICB*1(presence of insertion), and their associations with the highly polymorphic HLA-B locus were determined for 51 human cell lines and for 109 and 200 Caucasians and northeastern Thais, respectively. Analysis of the AluyMICB and HLA-B allelic relationships revealed that AluyMICB*1 occurred at relatively low gene frequency (0.118-0.157) [corrected] but was strongly associated with HLA-B17 (HLA-B57,HLA-B58) and HLA-B13. The AluyMICB locus provides a useful dimorphic marker for investigations on the level of linkage disequilibrium between MICB, MICA, and HLA-B loci.

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, and HLA-A29 cDNA clone was isolated and its predicted amino acid sequence compared with those of other cloned HLA class I genes. The isolated HLA-A29 cDNA corresponded to the rarer of the two A29 variant alleles, A29.1. Two amino acid residues of HLA-A29.1, gln-144 and arg-151, were found in all 24 HLA-B and HLA-C alleles examined but were present in only one of 15 HLA-A alleles for which sequence data are available. Importantly, this exceptional allele was HLA-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 the HLA-A alleles, but none of these was found in corresponding positions of HLA-B of -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-Aw19 cross-reactive group and the binding site of the monoclonal antibody 4E.

The loci for HLA-A,B,C,D, and DR are known to be closely linked to the structural loci for the complement components C2, BF, and the duplicated loci for C4, C4A and C4B. Conflicting evidence has been presented for the order of these genes. However, new techniques have made possible identification of markers in the HLA-D and C4 region for nearly all identified haplotypes. In our population we have confirmed five HLA-B-D crossovers and in each case informative allotypes of C2, BF, or C4A and C4B segregated with HLA-D or DR suggesting that the loci for these proteins lie close to HLA-D and DR. These findings may be of importance for resolving problems encountered in the assignment of HLA-D alleles.