Characterization of natural peptide ligands for HLA-B*4402 and -B*4403: implications for peptide involvement in allorecognition of a single amino acid change in the HLA-B44 heavy chain

Abstract: This study describes the characterization of endogenous peptides associated with the two major subtypes of HLA-B44. The two subtypes differ for a single amino acid substitution from Asp (HLA-B*4402) to Leu (HLA-B*4403) in position 156 of the α2 domain, causing strong alloreactivity in vivo. In order to study the involvement of peptides in this phenomenon, the peptide motifs of the two subtypes were determined from natural peptide pools using Edman degradation. The motif was found to be essentially identical for HLA-B*4402 and -B*4403, with a strong predominance for Glu at position 2, Tyr or Phe at positions 9 and 10 and hydrophobic residues, especially Met, at position 3. Two individual naturally processed ligands of HLA-B*4403 were sequenced and shown to be derived from intracellularly expressed proteins found in protein sequence databases. The sequence of these natural peptide ligands conform well to the determined motif. These data will allow the prediction of HLA-B44 restricted peptide epitopes from viral and tumor antigens of known amino acid sequences. Moreover, they indicate that the peptide repertoire presented by HLA-B*4402 and -B*4403 is very similar, suggesting that the strong alloresponse between these two subtypes is not due to presentation of a different set of self peptides.     

A tyrosinase nonapeptide presented by HLA-B44 is recognized on a human melanoma by autologous cytolytic T lymphocytes

The human tyrosinase gene has been reported previously to code for two distinct antigens recognized on HLA-A2 melanoma cells by autologous cytolytic T lymphocytes (CTL). By stimulating lymphocytes of melanoma patient MZ2 with a subclone of the tumor cell line of this patient, we obtained a CTL clone that lysed this subclone but did not lyse other subclones of the same melanoma cell line. The sensitive melanoma subclone was found to express a much higher level of tyrosinase than the others, suggesting that the antigen recognized by the CTL might be encoded by tyrosinase. Transfection of a tyrosinase cDNA demonstrated that the CTL clone indeed recognized a tyrosinase product presented by HLA-B*4403. The relevant antigenic peptide corresponds to residues 192–200 of the tyrosinase protein. Lymphoblastoid cells of the B*4402 subtype were not recognized by the CTL following incubation with the peptide. Nevertheless, by stimulating in vitro lymphocytes of a healthy HLA-B*4402 donor with autologous adherent cells pulsed with the same peptide, we obtained a CTL clone which recognized tumor cells expressing tyrosinase and HLA-B*4402. As HLA-B44 is expressed in 24% of Caucasians, the tyrosinase-B44 antigen may constitute a useful target for specific immunotherapy of melanoma.     

MOLECULAR TYPING AND HAPLOTYPIC ASSOCIATIONS OF HLA-B*44 SUBTYPES

The heterogeneity of HLA-B44 is confirmed and the sequence difference between the two major subtypes, B*4402,*4403, is attributed to one polymorphic site in the third exon. A method is described to discriminate B*4402 and B*4403, and the occurrence and linkage disequilibrium of B*44 subtypes is discussed. No example of B*4401 polymorphism in exon 2 was observed.     

HLA-B44 subtyping in the Catalan population using reference strand mediated conformation analysis. Implications for the selection of unrelated bone marrow donors

Cytotoxic T lymphocytes (CTLs) reactive against the disparity between HLA-B*4402 and HLA-B*4403 have been reported after unrelated donor bone marrow transplantation. These CTLs have been associated with acute graft-versus-host disease and graft rejection. This study describes the HLA-B44-subtyping in the Catalan population using reference-strand mediated conformation analysis. It has been performed on 297 unrelated HLA-B44+ cord blood units from the Barcelona Cord Blood Bank (Barcelona, Spain). We have found a predominance of HLA-B*4403 (66.04%) over HLA-B*4402 (33.02%), whereas the predominant HLA-B44 allele in Northern Europe and the United States is HLA-B*4402. This inverted proportion between HLA-B44 subtypes in Mediterranean populations compared with other Caucasian populations suggests that HLA-B44 subtyping should be performed when an HLA-B44+ unrelated donor marrow is identified.     

Relationship between peptide binding and T cell epitope selection: a study with subtypes of HLA-B27

The effect of HLA-B27 polymorphism on antigen presentation was analysed by comparing the binding of three Epstein-Barr virus-derived peptide epitopes to HLA-B27 subtypes with their immunogenicity and antigenicity in the context of these subtypes. The effect of altering the major anchor residue Arg2 on binding or on recognition by peptide-specific cytotoxic T lymphocytes (CTL) was also examined. The three peptides bound significantly to all the B*2701-B*2706 subtypes. This did not correlate with the peptides being immunogenic or recognized by specific CTL in the context of only particular subtypes. In addition, of the three viral epitopes tested, those that were immunogenic in B*2702- or B*2705-restricted responses bound to these subtypes less efficiently than one peptide that was immunogenic only in the B*2704 context. Thus, among several potentially immunogenic peptides from the same virus, the antiviral response is not necessarily directed against the one that binds best to the restricting subtype. These results indicate that HLA- B27 polymorphism influences antigen presentation in ways other than simply peptide affinity. Synthetic analogues lacking the canonical Arg2 motif of HLA-B27-bound peptides, even when binding much worse to the restricting subtype, were recognized equally by CTL specific for the parental peptide. This indicates that Arg2 is not required to maintain the structure of the epitope. The implications of these results for pathogenetic models of HLA-B27-associated disease are discussed.      

HLA-Cw*0409N is associated with HLA-A*2301 and HLA-B*4403-carrying haplotypes

The associations of HLA-B*4402 and HLA-B*4403 with alleles of HLA-A and HLA-Cw were investigated in panels of HLA-B*4403 and HLA-B*4402 homozygous individuals and in selected individuals carrying HLA-Cw*04 and HLA-B*4403. Some of these individuals were genotyped and also carried (HLA-DRB1*0701, DQB1*02). Among the latter, we studied individuals carrying the conserved extended haplotype (CEH) [HLA-Cw*04, B*4403, FC31, DRB1*0701, DQB1*02]. Four different common (HLA-Cw*, B*44) haplotypes were identified that extended to the HLA-A locus: HLA-A*0201, Cw*0501, B*4402; HLA-A*2902, Cw*1601, B*4403; HLA-A*2301, Cw*0401, B*4403; and HLA-A*2301, Cw*0409N, B*4403. We identified eight unrelated examples of the allele HLA-Cw*0409N. HLA-A*2301 was associated with both HLA-Cw*0401 and HLA-Cw*0409N, suggesting that HLA-Cw*0409N may have arisen from a mutation in a CEH. We estimate that approximately 2 to 5 in 1000 Caucasian individuals carry the allele HLA-Cw*0409N, making it one of the most frequent null HLA alleles known to date. Our findings demonstrate the first example of three different HLA-Cw-determined subtypes of a common or CEH carrying a shared HLA-B allele, in this case HLA-B*4403.     

Complete HLA-B44 subtyping in Spaniards. Additional evidence for heterogeneity in Caucasian populations

HLA-B44 is one of the most common HLA class I alleles in Caucasians. Exon 3 oligotyping and sequence analysis have define five B44 subtypes: B^*4402, B^*4403, B^*4404, B^*4405 and B^*4406, with variations in exons 2 and 3. We have developed a conventional DNA typing system by using a single B12-group specific amplification including exon2-intron2-exon3 in combination with 6 oligoprobes to define all B44 subtypes. 140 HLA-B44 positive unrelated Spanish Caucasians were typed. Family studies established 30 B44-bearing haplotypes. The distribution of B44 subtypes in our population was: B^*4402 32.5%, B^*4403 66.5%, B^*4404 0.5%, B^*4405 0.5%, B^*4406 not found. B^*4402 and B^*4403 represented the 99% of the B44 alleles, as described in Caucasians. However, these two major subtypes showed an inverted frequency when compared with other Caucasian populations, B^*4403 twice as frequently as B^*4402 in Spaniards. HLA-B44-associated chromosomes showed 20 different haplotypes (including HLA-A,-C,-DR,-DQ), although demonstrating clear separated haplotype composition between B^*4402 and B^*4403: B^*4402 associated to class I alleles A2 ( ) and Cw5 ( ), and B^*4403 associated to the class II allele DRB1^*0701 ( ). These findings, in addition to the validation of a complete B44 oligotyping system, revealed further evidence of antigen frequency differences among populations of the same ethnic origin.     

HLA-B44 ALLELE FREQUENCIES AND HAPLOTYPIC ASSOCIATIONS IN THREE EUROPEAN POPULATIONS

HLA-B44 is among the most frequent class I antigens in many populations studied so far. It has been subdivided into seven allelic forms that can only be discriminated by DNA typing. Using a simple PCR/sequence-specific oligonucleotide hybridization procedure, we have analysed the frequency distribution of B44 subtypes in three European populations from Slovenia, the Netherlands, and Switzerland. B*4402 and B*4403 were by far the predominant alleles, B*4404 and 4405 were rare, while B*4406 and B*4407 were not observed. Interestingly, B*4402 and 4403 occurred with different frequencies in the three populations, with B*4402 being most frequent in the Swiss (65% vs. 57% in the Dutch, and 46.5% in the Slovenes). Of the 139 individuals studied, 60 HLA-B44 ABDR haplotypes could be determined by family studies. In the respective populations, the linkage disequilibria between B44 and other HLA antigens occurred with different frequencies. A2-B*4402 haplotypes were very frequent in the Swiss sample, mostly associated with DRB1*0101, 0401 and 1301. B*4402 was more often linked with non-A2 antigens in the Slovenes (predominantly A24, A28) than in the Swiss and the Dutch. The predominant association of B*4403 was with DR7: this haplotype was very frequent in the Swiss (82% of the B*4403 haplotypes), while lower frequencies were found in the Dutch (72%) and Slovenian (59%) populations. In the Swiss population, more than half of the B44-DR7 haplotypes were A23-B*4403-DR7 (53% of all B*4403 haplotypes). This haplotype was significantly less frequent in the Slovenian (6%) and in the Dutch (14%) populations. The second most frequent B*4403 haplotype in both the Swiss and Slovenes is the A29-B*4403-Cw*1601-DR7 haplotype (17.6 and 29.4%, respectively). Concomitant with the increased frequency of B*4403 in the Slovenes, a higher diversity of non-DR7 B44 haplotypes was observed in this population (41% of all B*4403 haplotypes). HLA-B44 oligotyping analysis allowed us to detect B44-subtype incompatibilities in several AB-sero, DRB1/B3/B5-oligo matched unrelated bone marrow donor/patient combinations. The different frequency distributions of HLA-B44 haplotypes in the three populations analysed in this study argue in favour of local volunteer bone marrow donor recruitment. This might significantly improve the chance of finding a highly matched donor for patients with less frequent A-B-DRB1 haplotypes.     

HLA-B*44 allele frequencies and haplotypic associations in Koreans

We have investigated the frequencies of HLA-B*44 alleles and their haplotypic associations with HLA-A, -C, and -DRB1 loci in 450 healthy unrelated Koreans, including 213 parents from 107 families. All 79 samples (17.6%) typed as B44 by serology were analyzed for B*44 alleles using polymerase chain reaction (PCR) single-strand conformation polymorphism (SSCP) method. A total of three different B*44 alleles were detected: B*44031 (allele frequency 4.7%), B*44032 (3.1%), and B*4402 (1.3%). Three characteristic haplotypes revealing strong linkage disequilibrium were A*3303-Cw*1403-B*44031-DRB1*1302 (3.6%), A*3303-Cw*07-B*44032-DRB1*0701 (2.8%), and A*3201-Cw*05-B*4402-DRB1*0405 (0.4%). In addition, a strong association was observed for B*4402 with A*0301. The B*4403-bearing haplotypes of Koreans appear to be relatively common in Asian populations, whereas the B*4402-bearing haplotypes share some similarity to those of Caucasians. HLA-B44 alleles demonstrate a limited allelic diversity and comprise distinctive extended haplotypes in the Korean population. It is suggested that the frequencies of B44 subtype mismatches among ABDRB1-matched unrelated donor-recipient pairs would be low in this population.     

Inefficient exogenous loading of a tapasin-dependent peptide onto HLA-B*44:02 can be improved by acid treatment or fixation of target cells

Antitumor cytolytic T lymphocytes (CTLs) recognize peptides derived from cellular proteins and presented on MHC class I. One category of peptides recognized by these CTLs is derived from proteins encoded by "cancer-germline" genes, which are specifically expressed in tumors, and therefore represent optimal targets for cancer immunotherapy. Here, we identify an antigenic peptide, which is derived from the MAGE-A1-encoded protein (160-169) and presented to CTLs by HLA-B*44:02. Although this peptide is encoded by MAGE-A1, processed endogenously and presented by tumor cells, the corresponding synthetic peptide is hardly able to sensitize target cells to CTL recognition when pulsed exogenously. Endogenous processing and presentation of this peptide is strictly dependent on the presence of tapasin, which is believed to help peptide loading by stabilizing a peptide-receptive form of HLA-B*44:02. Exogenous loading of the peptide can be dramatically improved by paraformaldehyde fixation of surface molecules or by peptide loading at acidic pH. Either strategy allows efficient exogenous loading of the peptide, presumably by generating or stabilizing a peptide-receptive, empty conformation of the HLA. Altogether, our results indicate a potential drawback of short peptide-based vaccination strategies and offer possible solutions regarding the use of problematic epitopes such as the one described here.     

Analysis of HLA-B*44 alleles encoded on extended HLA haplotypes by direct automated sequencing

Abstract: We developed a PCR-based approach to sequence exons 2 and 3 of HLA-B44 alleles from genomic DNA. We applied this method to determine the B44 alleles encoded on extended HLA-A, B, DRB1, DQB1 haplotypes and the degree of mismatching for B44 alleles among marrow transplant patients and their unrelated donors (URD). A total of 81 samples was studied and included 38 patients, 42 donors and the cell "FMB"; the 80 clinical samples were comprised of 8 unpaired patients, 12 unpaired donors, and 30 URD-recipient pairs. Three alleles encoding B44 were identified, B*4402 (N=51), 4403 (N=32) and a new allele designated B*44KB and named B*4405 (N=4). Of the 27 patients for whom family study was available, there were 13 different B*4402, 7 different B* 4403 and 2 new B*4405 haplotypes. HLA-A2, Cw*0501, B*4402, DRB1* 0401, DQB1*0301 (n=2); A2, Cw*0501, B*4402, DRB1*1501, DRB5* 0101, DQB1*0602 (n=2); and HLA-A29, Cw*1601, B*4403, DRB1* 0701, DQB1*0201 (n=5) comprised the most common patient haplotypes. Of 30 URD-recipient transplant pairs studied, 27 were HLA-A, B serologically matched and DRB1, DRB3, DRB5, DQB1 allele matched, and 3 pairs were DRB1-mismatched. All B44 allele mismatching (N=3) occurred among the 27 matched pairs. The novel B*4402-variant sequence, HLA-B*4405, was identified in 4 individuals, and in each case was associated with an HLA-B44, Cw*02022, DRB1*0101, DQB1*0501 haplotype. HLA-B*4405 and B*4402 are identical in exon 2; in exon 3 however, B*4405 encodes T instead of G at nucleotide position 75 which translates to a substitution of tyrosine for aspartic acid at codon 116. Finally, the published B*4402 sequence derived from cell "FMB" was found to contain an error; the corrected B*4402 sequence encodes G rather than C at position 146 of exon 3.     

HLA-B44 subtyping in a Spanish population: further evidence of Caucasian population diversity

HLA-B44 is the most frequent HLA-B allele in Caucasian populations. Several B44 subtypes, B*4402-B*4406, have been identified in individuals with this ethnic origin. Mismatches among B44 subtypes have been described as major targets for allogeneic responses in bone marrow transplantation. We have developed a PCR-SSO method, based on a B12- specific DNA amplification of exon 2 through exon 3 and subsequent non radioactive hybridization with eight probes, which allow us to discriminate all B12 homozygous combinations. We applied this method to determine the frequency of B44 subtypes in a Spanish population, as well as their HLA-A.-C.-DRB1,-DRB3/DRB4/DRB5.-DQA1 and -DQB1 associated haplotypes. A total of 141 healthy unrelated Spanish individuals and 31 B44-bearing haplotypes were investigated. Four B44 alleles were identified, B*4402 (33%), B*4403 (66%), B*4404 (0.7%), and B*4405 (0.7%). Haplotype analysis showed a clear differentiated distribution pattern for the two major B44 subtypes. B*4402 is associated with Cw5 (11/13) and A2 antigens (10/13). In contrast, B*4403 is mainly found together with DRB1*0701 (14/16). An inverted B*4402/B*4403 frequency in comparison with other European and North American Caucasian populations, revealed the existence of an extended haplotype diversity between populations of the same ethnic origin. Apart from anthropological studies, high resolution typing for HLA class I antigens presenting molecular polymorphism will be of great relevance in unrelated bone marrow transplantation.     

T-cell receptor usage in alloreactivity against HLA-B*2703 reveals significant conservation of the antigenic structure of B*2705

B*2703 is an exceptional HLA-B27 molecule in that it differs from the most common B*2705 subtype by a unique amino acid change (His59) altering N-terminal peptide anchorage. To assess how this unusual feature affects the antigenic structure of HLA-B27, TCR usage by alloreactive CTL raised against B*2703 from two individuals was analyzed. Only few CTL recognized B*2703 but not or at a lower level B*2705. Limited heterogeneity of these CTL was revealed by: 1) identity of TCR in two pairs of such CTL clones, 2) identity of β chains, paired to distinct α chains, in two clonotypes, and 3) almost identical fine specificity of these two clonotypes with site-specific HLA-B27 mutants. These results indicate that B*2703 "private" epitopes are rare. TCR usage among anti-B*2703 CTL was analogous as in anti-B*2705 responses in the predominant and donor-independent usage of Vβ segments from homology subgroup 4, more moderate and donor-dependent Vα skewing, N+Dβ diversity limited by motifs shared among clonotypes, and restricted Jα heterogeneity. Homology of N+Dβ motifs and Jα segments of anti-B*2703 with anti-B*2705 TCR suggested significant sharing of peptide-associated epitopes between both subtypes. The results indicate that allospecific TCR are recruited by B*2703 following similar rules as in the anti-B*2705 response, and suggest that the B*2703 change keeps unaltered much of the antigenic structure of the molecule relative to B*2705. Therefore, most of the peptides bound to B*2703 should be the same and keep a similar conformation as in B*2705.     

A MAGE-3 peptide presented by HLA-B44 is also recognized by cytolytic T lymphocytes on HLA-B18

Antigens encoded by MAGE genes are of particular interest for cancer immunotherapy because of their tumoral specificity and because they are shared by many tumors. Antigenic peptide MEVDPIGHLY, which is encoded by MAGE-3 and is known to be presented by human leukocyte antigen (HLA)-B44, is currently being used in therapeutic vaccination trials. We report here that a cytolytic T lymphocyte (CTL) clone, which is restricted by HLA-B*1801, recognizes the same peptide and, importantly, lyzes HLA-B18 tumor cells expressing MAGE-3. These results imply that the use of peptide MEVDPIGHLY can now be extended to HLA-B18 patients. We also provide evidence that, under limiting amounts of protein MAGE-3, HLA B*1801 and B*4403 compete for binding to the peptide.     

An HLA-B27 polymorphism (B*2710) that is critical for T-cell recognition has limited effects on peptide specificity

Abstract: The B*2710 subtype differs from the HLA-B27 prototype (B*2705) only by having Glu instead of Val at position 152, in the α2 helix of the peptide-binding site. In spite of its structural similarity most allore-active CTL raised against B*2705 fail to cross-react with B*2710. Indeed, of the residues that are polymorphic among HLA-B27 subtypes, the Val> Glu152 change has the greatest influence on HLA-B27 T-cell antigenicity. The molecular basis for this antigenic disparity was analyzed in this study. Sequence analysis indicated that B*2710-bound peptides have very similar motifs to B*2705-bound ones both at the main and auxiliary anchor positions. In addition, most of the individual ligands sequenced from B*2710 were previously found in B*2705. Together these results indicate that both subtypes have largely overlapping peptide repertoires. Molecular dynamics simulations of a common ligand in complex with either B*2710 or B*2705 failed to detect significant conformational changes in the peptidic main chain or in solvent accessibility of the side chains. In addition, modeling of the Val>Glu152 change into the MHC-peptide-TCR structure suggested a direct role of residue 152 in interaction with the TCR. Thus, the large differences in T-cell recognition between B*2710 and B*2705 are not explained by an effect of the Glu152 change on peptide specificity or conformation, but by different direct interactions with the TCR.     

Identification of multiple HIV-1 CTL epitopes presented by HLA-B*5101 molecules.

We attempted to identify and characterize HIV-1 CTL epitopes presented by HLA-B51 which is associated with a slow progression to AIDS. HLA-B*5101 stabilization assay showed that 33 out of 172 HIV-1 peptides carrying HLA-B*5101 anchor residues bound to HLA-B*5101. Seven peptides were suggested as HIV-1 CTL epitopes presented by HLA-B*5101 because the specific CTL was induced for these peptides in PBMC from three HIV-1 seropositive individuals carrying HLA-B51 by stimulation with HLA-B*5101 binding peptides. Analysis of these epitopes using the specific CTL clones confirmed that six of seven HIV-1 peptides are epitopes presented by HLA-B*5101. Three epitopes presented by HLA-B*5101 are highly conserved among the clade B strain, suggesting that the specific CTL for these epitopes might play an important role in recognition of HIV-1 infected cells. These epitopes will be useful to analyze CTL responses in HIV-1 infected individuals.     

Susceptibility to ankylosing spondylitis correlates with the C-terminal residue of peptides presented by various HLA-B27 subtypes

Susceptibility to spondyloarthropaties is strongly associated with some HLA-B27 alleles. Evidence suggests a direct pathogenic role for the B27 molecules which possibly present an arthritogenic peptide to the T cells. If this hypothesis is true, B27 subtypes that differ structurally but are disease-associated ought to be capable of presenting such peptide(s), while non-disease-associated ones would not. We have recently described a B27 subtype, B*2709, and shown its absence in ankylosing spondylitis (AS) patients. Here, we show the elution and sequence of peptides from HLA-B*2709 molecules. Similar to other B27 subtypes, these peptides are mainly nonamers with an Arg at position P2. Comparison of the C-terminal anchors of peptides eluted from B*2702 and B*2705 with those eluted from B*2709 reveals that, while B*2702 and B*2705 have a broader specificity, B*2709 molecules appear to only accept C-terminal hydrophobic residues. A common feature shared by the two caucasoid AS-associated subtypes (B*2702 and B*2705) but different from B*2709, is the presence of a Tyr as peptide C-terminal anchor. The substitution of Val for Tyr at the C terminus in one of the eluted peptides greatly reduces the binding to B*2709 molecules. This finding suggests Tyr as a discriminative amino acid allowed at the C terminus of peptides bound to the AS-associated B27 subtypes, but not to those which are not associated with AS.     

Analysis of 250 HLA-B44 genotypes in European Caucasoids: high diversity and preferential ABCDRB1 associations in B*4402, B*4403, and B*4405 haplotypes

Based on high-resolution DNA typing within 235 pedigrees, a total of 250 HLA-A/B/C/DRB1/DRB3 genotypes have been characterized. These comprise 129 different B44 haplotypes, of which 73.6% occurred only once. Only four different B*44 alleles were identified: B*4402-4405, with B*4402 and B*4403 haplotypes accounting for 57.6 and 36.8%, respectively, of all haplotypes. Although the relative numbers of different A/B/C/DRB1/B3 haplotypic associations were similar in both B*4402 and B*4403 haplotypes, the genotypic profiles were quite different in the two groups. When associated with the A*0101, A*0201, A*2402, A*3201, and A*6801 alleles, a much more extensive polymorphism of B*4402 haplotypes with respect to HLA-C and DRB1 associations was disclosed. On the other hand, B*4403 haplotypes were more diverse in the A23-B44 and A29-B44 groups with respect to DRB1 associations. Considering B-C linkage, B*4402-Cw*0501, B*4402-Cw*0704, B*4402-Cw*1604, B*4403-Cw*0401, B*4403-Cw*1601, B*4404-Cw*1601, and B*4405-Cw*0202 accounted for 98% of all genotypes. Eight A/B/C/DRB1 haplotypes occurred at a relative genotypic frequency of >0.015, with A*2902-B*4403-Cw*1601-DRB1*0701 (11.2%) and A*0201-B*4402-Cw*0501-DRB1*0401 (8.4%) as the two most frequent genotypes. Some A and DRB1 alleles were predominantly, if not exclusively, associated with specific B-C pairs: A*0301 with B*4402-Cw*0501 and B*4403-Cw*0401; A*2301 with B*4403-Cw*0401; A*2608 with B*4402-Cw*0501; A*2902 with B*4403-Cw*1601; DRB1*0101/0401/0403/0404/1101/1104/0801/1301/1302 with B*4402-Cw*0501; and DRB1*0701 with B*4403-Cw*1601. On the basis of this dataset and our experience with searches for phenotypically matched unrelated stem cell donors, several ABDR haplotypes were identified that would confer a higher probability of B44- and C-incompatibility. The analysis of 112 consecutive unrelated stem cell donor searches revealed that 24% of the 400 tested donors were B44-mismatched, and that no single B44 allele- matched donor could be identified for only 7% of the patients. HLA-C incompatibility rate was 22.2% for the patients with > or =1 B44 allele-matched donor(s). This dataset can therefore be used as a predictive tool for B44- and C-disparities in unrelated stem cell transplantation.     

A new HLA-B44 variant (B44BO [B*4408]) identified by serology

Using HLA serology, we detected a new variant of HLA-B44 — B44BO — in two families. This antigen reacts with B44 antisera and is negative with over one-third of B12 (B44, B45) sera but reacts with 50% of antisera with a B62 component, especially if they contain anti-B57. The variant, B*4408, differs from the common B*4402 by 4 nucleotide substitutions in exon 2: 193, 206 and 209, which produce changes in the α1domain at positions 41, 45 and 46 (TKE in B*4402 and AMA in B44BO); and nucleotide 213, a silent substitution. At each of these positions, B*4408 is identical to B*46, B*57 and many B*15 alleles. As anticipated from its predicted iso-electric point (5.71), one-dimensional isoelectric focusing studies showed that B44BO focuses at the same position as B*4402. The sequence and serological reactivity of this rare antigen allowed the identification of two likely epitopes shared by two different groups of HLA-B antigens.     

Subtypes of B44 in six different populations.

We have investigated the distribution of HLA-B44 subtypes in various populations, see table. Of the five B44 subtypes investigated, two are apparently quite rare because they were only found in the local central european panel (B^*4404 and B^*4406). The european populations are characterized by a relatively high frequency of B^*4402, while in the Albanian, in the Asiatic and African populations, the subtype B^*4403 is prevailing. The distribution of the B44 subtypes in the Czech and the Munich population are virtually the same. We conclude from these data, that B44 subtypes have different distributions in different populations of the world and must therefore be taken into consideration when matching for bone marrow transplantation.