Nucleotide sequences of novel HLA-B35 subtypes

In recent reports we described novel hybridization patterns (HP) corresponding to 22 potentially new HLA-B locus alleles in a panel of 547 subjects studied by PCR-SSOP. Three of them correspond to new subtypes of B35. To confirm the hybridization results we have isolated DNA from PBL and performed PCR, DNA cloning and nucleotide sequencing. One of the alleles, locally called B-3505v was found in three individuals: two Hispanic, one Caucasoid. It differs from B^*3505 by 3 nucleotide substitutions that lead to changes in residues 94 (Ile > Thr), 95 (Ile > Leu) and 103 (Val > Leu). B-3505v differs from B^*3501 in residues 97 and 103. Another allele called B-3508v, was found in 7 individuals, (6 of 122 Toba Indians, 1 of 18 Pilaga Indians). It differs from B^*3509 in two silent nucleotide substitutions (codons 135 and 138) and in one substitution in residue 156 (Arg > Leu). The new allele has a hybrid sequence between B^* 3508 and B^*4801. A third subtype, locally called B-3504v, observed in two Hispanic individuals, is identical to B^*3512. B^*3512 differs from B^*3504 by 3 nucleotides and one amino acid. Substitutions in residue 95 contribute to the structure of specificity pocket F, 97 to C and E, and 156 to pockets D and E. Therefore it is possible that some of the new alleles may have different peptide binding profiles. Since differences in residue 156 have been shown to affect allorecognition and mediate GvHD, identification of such variants may have important implications in transplantation and perhaps in studies of immune responses to peptides and pathogens.     

The relative distribution of B35 alleles and their IEF isotypes in a HLA-B35-positive population

Abstract: The HLA-B35 serotype represents a group of antigens detectable by IEF, cytotoxic T cells, and by sequencing analysis. Four isotypes and eight alleles have been thus far reported. We have determined the relative frequencies of these B35 subtypes in a group of 203 unrelated people. Dot blot hybridization of PCR amplified products was performed using 23 sequence-specific oligo probes designed based on the EMBL HLA class I sequence database. The amplification was achieved by a pair of group-specific primers, producing ˜600 bp fragments. By hybridization pattern analysis, we found that four alleles represent over 95% of the B35+ population, with relative frequency of 48.2% for B*3501, 23.7% for B*3502, 15.2% for B*3503, and 8.0% for B*3508. We also identified 3 individuals with B*3504 and one with B*3505, and seven samples with new patterns. B*3501 and B*3503 exactly correlated with the most common isotype B35.3, B*3502 and B*3504 with B35.2, B*3508 may be the B35.1 IEF isotype. The B*3505 was identified from an individual with B35 IEF variant form. Our study shows that the B35 antigen has a wide distribution of alleles, and that many more B35-related alleles may yet to be uncovered.     

Allelic heterogeneity of HLA-B35 subtypes in different populations as assessed by DNA typing

Abstract: HLA-B35, a class I antigen differentially associated to several diseases in different ethnic groups, comprises at least eight alleles which differ among them by one to six amino acids. In the present work a rapid DNA typing procedure was used to investigate the distribution of the various HLA-B35 alleles in different populations. The approach is based on a group-specific PCR amplification of a set of closely related HLA-B alleles sharing a Thr in position 45 of the alpha-1 domain. The amplified DNA was then hybridized to a panel of sequence-specific oligonucleotide (SSO) probes designed to recognize the polymorphic residues in previously reported HLA-B35 subtypes. This methodology was successfully tested in 100 individuals of four different populations, previously typed by serology as HLA-B35, and in six reference panel cells of the 10th International Histocompatibility Workshop. HLA-B*3501 was the predominant subtype in all populations. B*3502, B*3503 and, to a lesser extent B*3508, were also found. Among Mexican Mestizos, thirteen individuals had patterns of SSO hybridization suggestive of new B35 alleles. The evolutionary considerations on the different B35 alleles and their extended B35, Cw4 haplotypes are discussed.     

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-B40, B18, B27, and B37 allele discrimination using group-specific amplification and SSCP method

We developed a system for discriminating HLA-B40, B18, B27, and B37 alleles using a two-step PCR method followed by SSCP analysis. Fragments (0.8 kb) including exon 2, intron 2, and exon 3 were amplified in the first PCR. We used two sets of primers, one specific for HLA-B60-related alleles and the other specific for HLA-B6l-related, B18, B27, and B37 alleles. No amplifications of other class I genes or pseudogenes were observed. In the second PCR, exon 2 and exon 3 were amplified separately, using diluents of the first PCR products as templates. HLA-B6l-related, B18, B27, B37, and B60-related alleles were clearly discriminated in the SSCP analysis of the second PCR products. In a population study in which B6l alleles were analyzed, B^*4003 was detected in two Japanese individuals in addition to two B6l alleles previously reported to occur in Japanese, B^*4002 and B^*4006. The relative frequencies of B^*4002, B^*4006, and B^*4003 in Japanese were 58, 35, and 6%, respectively. The individuals having B^*4003 are the first non-South Americans in whom this allele has been detected. The SSCP banding patterns of 18 HLA-B60-positive Japanese population samples were identical to those of a B^*40012 sample for both exon 2 and exon 3. We also demonstrated that the B37 allele occurring in some Japanese is B^*3701.     

New alleles of HLA-B35 in South India.

Serological pattern analysis of HLA B35 alleles from south India revealed the existence of polymorphism in the HLA-B35 alleles. HLA-B typing was carried out on 42 HLA-B35 individuals from south India using 210 antisera of AHS#4, XII workshop: all these cells were those characterized in III AOH and XI IHW. Of the 42 cells analysed for HLA-B35 sub-types by PCR-SSOP technique, 3 alleles predominated; HLA-B*3503 followed by B*3501/5 and B*3501/5/8. These were correlated to serological patterns of the cells typed in XI and XII workshops. The analysis identified some sera which did not identify HLA-B*3503 and B*3501/5. The serological patterns of HLA-B*3501/5/8 and B*3503 were clearly assignable in Iyer caste group by sera WEG 107 & HA3 109 sera of XI IHWC but not in many south Indian samples. A systematic analysis of B*35 in various Indian caste groups is warranted.     

Conservation of genes encoding HLA-B5 and B35 cross-reactive group antigens in various races

HLA-B5 and B35 CREG antigens include HLA-B35, B51, B52, B53, and B78. Recent studies suggest that the genes encoding the HLA-B5, B35 CREG, and HLA-B58 antigens share a common ancestor. We sequenced the exons of the genes encoding HLA-B51, B53, and B58 from American black individuals and the gene HLA-B52 encoding from an Arabic individual, and compared them with previously reported sequences of HLA-B51 (B^*5101) and HLA-B52 (B^*5201) from Japanese, HLA-B53 (B^*5301) from an Algerian, and HLA-B58 (B^*5801) from a Sardinian. The sequences of the genes from the American black and Arabic individuals were identical to those from the other races. These findings support the hypothesis that these antigens have evolved prior to divergence of the major ethnic groups.     

HLA-B27 subtypes in chinese patients with ankylosing spondylitis

The HLA-B27 allele has been extensively studied due to its strong association with ankylosing spondylitis (AS). In order to identify B27 alleles in Chinese patients with AS from the Shanghai area, we joined the AHS#5 of the 12 IHW and total of 68 B27 positive patients and 7 B27 positive normal persons have been investigated using polymerase chain reaction and Dig-ddTUP labeled oligonucleotides. Three primer pairs, E403 and E90as, E91As and E136as, E91Bs and E18as, were used to amplify codons 40-90 of HLA-B related alleles, codon 91 to 136 of HLA-B^*2701-B^*2706 and B^*2708 and codons 91-180 of B^*2707. A total of 11 probes were used to distinguish 8 B^*27 alleles from B^*2701 to B^*2708. 68 AS patients contain 69 B27 alleles because one patient is heterozygous B^*2704/B^*2705. A total of 4 alleles of B^*27 were detected in the AS-patient group. B^*2704 is the most common B^*27 allele in both AS patients and controls with similar frequency, 76.8% and 71.4%, respectively. We found a high proportion of B^*2705 in both AS patient (20.3%) and control (28.6%) groups. Although the control group is quite small we are still able to deduce that B^*2704 and probably also B^*2705 seem to be associated with AS in Shanghai area patients We also found one AS allele typed as B^*2707. Interestingly, for the first time we detected B^*2706 in an AS patient, which would argue against a protective effect of B^*2706 on AS susceptibility in Shanghai Chinese. The conclusion from this study is that the distribution of B^*27 alleles is not significantly different between AS patients and controls. Expanded numbers of AS patients and especially of healthy controls in different ethnic groups will be necessary to assess the contribution of different B27 subtypes to AS susceptibility.     

Microheterogeneity in HLA-B35 alleles influences peptide-dependent allorecognition by cytotoxic T cells but not binding of a peptide-restricted monoclonal antibody

Strong peptide dependency of HLA-B^*3501-specific alloreactive T-cell clones was observed in the recognition of cells bearing closely related B35 variants. The single amino acid exchange in the ß-pleated sheet of B*3503 completely abolished the responses of all clones, whereas an amino acid exchange in the ₂ helix of the newest B35 member (B*3508) only altered allorecognition of one T-cell clone, demonstrating the differential impact of these positions on peptide binding to B35 molecules. In contrast to T cells, amAb (TÜ165) recognizing the B35 specificity in a peptide-dependent manner bound to the B35 variants irrespective of their sequence heterogeneity. However, quantitative binding differences were detected with cells bearing the same B35 alleles. This is most likely due to variations in the amount of peptide(s) that associates with B35 and forms the ligand seen by this mAb. These results reveal how naturally occurring single amino acid substitutions have led to generation of functionally distinct molecules of another multimember HLA class I cluster.     

Two new alleles of HLA-B22 group in Japanese

Five different B22 split antigens, B54, B55.1, B55.2, B56 and B22N, have been reported in Japanese. Genomic DNAs were analyzed by PCR-SSCP and sequencing methods for Japanese samples serologically positive for each split antigen. In the SSCP analysis of exon 2, B55.1, B55.2 and B56 showed identical patterns, whereas B54 and B22N showed distinct patterns. In the SSCP analysis of exon 3, B54 and B55.1 showed identical patterns, whereas B55.2, B56 and B22N showed distinct patterns. Sequence analysis further showed that B54, B55.1 and B56 were encoded by B^*5401, B^*5502 and B^*5601, respectively. The other two rare antigens, B55.2 and B22N, were found to be encoded by new alleles. The exon 2 amino acid sequences encoded by B55.2 and B22N alleles were identical to those of B55- and B56-group alleles, although that of B22N included a synonymous substitution. The exon 3 sequence of B55.2 was unique among HLA-B alleles. The exon 3 sequence of B22N was identical to those of B^*1501 and B^*4601, but distinct from those of other B22-group alleles.     

Characterization of a new HLA-B allele (B3702) generated by an intronic recombination event

Routine serological HLA typing of a Syrian family revealed a Bw4-associated HLA-B blank antigen showing Mendelian segregation together with the haplotype A1, Cw2, DR11, DQ7. A more extensive serological analysis showed no conclusive typing reactions with sera towards HLA-B27 and HLA-B37 antigens. Full length cDNA was sequenced in both senses for two individuals. Comparison of the consensus sequence with all other HLA-B published sequences evidenced a new HLA-B allele, confirming, as serologically predicted, the greatest relationship to HLA-B37 and HLA-B27 genes. Based on the exon 2 sequence identity to B^*3701, this new allele has been designated B^*3702. Proteins derived from B^*3702 and B^*3701 differ in 9 amino acids at alpha 2 (7 residues), and in transmembrane and cytoplasmic domains. Exons 3 to 8 are identical between B^*3702 and a number of HLA-B27 subtypes (all except Oriental HLA-B27 subtypes B^*2704, B^*2706 and B^*2707). B^*3702 alpha 1 domain differs from those of B^*2701, B^*2702, B^*2703, B^*2705, and B^*2708 in 9, 11, 9, 7, and 11 residues, respectively. The mosaicism found in B^*3702 strongly suggests that this new allele could be derived by homologous recombination at the intron 2 region between the B^*3701 gene and some of the 5 non-oriental B27 subtypes.     

Two new class I alleles (HLA-Cw15052 and B0706) constitute a novel haplotype in Caucasians.

HLA-Cw^*15 alleles have been previously described in association with the HLA-B specificities B51, B61 and B73. In this study, we have characterized a novel haplotype constituted by the new alleles Cw^*15052 and B^*0706.

The two variants of Cw^*1505 differ in a synonymous change in nucleotide 24 of exon 1 coding for the signal peptide. The new B^*0706 allele differs from B^*0702 and B^*0705 in single changes at exons 3 and 5, respectively, and could be considered an intermediate evolutionary step between them.

Even though strong linkage disequilibrium between the HLA antigens Cw7 and B7 has been observed, ‘HLA-C blank’-B7 haplotypes are not rare either. Cw^*15 and B7 have been found to be associated in several Caucasian individuals. Therefore, it is likely that Cw^*15 accounts for a part of the aforementioned ‘HLA-C blank’-B7 haplotypes.

With the identification of Cw^*15052, a previously proposed pathway for the evolution of Cw^*15 alleles can be completed and will be discussed.     

Generation of cytotoxic T cell responses directed to human leucocyte antigen Class I restricted epitopes from the Aspergillus f16 allergen

Invasive aspergillosis (IA) is a major cause of infection-related mortality in patients with haematological malignancies, especially in recipients of haematopoietic stem cell transplants. We have prepared overlapping pentadecapeptides (11-aa overlap with previous peptide) spanning the entire 427-aa coding region of the Aspergillus allergen, Asp f16 shown previously in mice to induce Th1-type cell responses in vivo and in humans to induce proliferative and cytotoxic CD4(+) T cell responses. Mature dendritic cells (DC) pulsed with a complete pool of peptides were used to generate T cell lines. Two lines from HLA-B*3501(+) donors were found to be strongly cytotoxic to autologous Asp f16-peptide pool- and Aspergillus culture extract-pulsed targets after 4-5 weekly primings. Cytotoxic T lymphocyte (CTL) culture supernatant killed Aspergillus conidia, and cells directly killed Aspergillus hyphae. Cytotoxic activity and interferon (IFN)-gamma production were mediated exclusively by CD8(+) T cells in response to pool-pulsed targets. Interleukin (IL)-4 production was not detected. CTL activity was restricted by HLA-B*3501 and based on peptide prediction programmes was most probably directed to YFKYTAAAL (YFK), LPLCSAQTW (LPL) and GTRFPQTPM (GTR) in one donor, while only LPL was recognized by CTL from the second donor. Pool-pulsed B*3503(+) BLCL but not B*3502(+) or B*3508(+) BLCL presented peptide to donor no. 1. B*3503(+) BLCL presented YFK and to a lesser extent GTR, but not peptide LPL. Our data show that in addition to our previously identified Class II restricted peptide response, DC pulsed with a pentadecapeptide pool from Asp f16 are capable of inducing polyclonal, HLA-Class I-restricted, Aspergillus-specific T cells that may be capable of conferring immunity to IA.     

Identification of the gene encoding a novel HLA-B39 subtype Two amino acid substitutions on the β-sheet out of the peptide-binding floor form a novel serological epitope

Serologic analysis suggests the existence of a novel HLA-B39 subtype (HLA-B39N) in the Japanese population. To identify this novel allele, a gene encoding HLA-B39N was cloned and the exons were sequenced. A gene encoding HLA-B39N (B^*3904) and B^*39011 differs by two nucleotide substitutions at codons 11 and 12 whereas B^*3904 and B^*39013 differ by three nucleotide substitutions at codons 11, 12, and 312. One nucleotide difference at codon 11 produces a change from serine in B^*3901 to alanine in B^*3904 whereas another difference at codon 12 changes valine in B^*3901 to methionine in B^*3904. The residues 11 and 12 are located on the β-sheet out of the peptide-binding floor and are completely buried in the molecule. These results suggest that the substitutions at these residues alter the conformation of other residues forming epitopes of alloantibodies. Analysis of HLA-B^*3901 genes in the Japanese population showed that both B^*39011 and B^*39013 were observed in the Japanese population. The present study suggests that B^*3904 may have evolved from B^*39011 rather than B^*39013.     

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.     

The impact of HLA-B micropolymorphism outside primary peptide anchor pockets on the CTL response to CMV

The factors controlling epitope selection in the T cell response to persistent viruses are not fully understood, and we have examined this issue in the context of four HLA-B*35-binding peptides from the pp65 antigen of human cytomegalovirus, two of which are previously undescribed. Striking differences in the hierarchy of immunodominance between these four epitopes were observed in healthy virus carriers expressing HLA-B*3501 versus B*3508, two HLA-B allotypes that differ by a single amino acid at position 156 (HLA-B*3501, (156)Leucine; HLA-B*3508, (156)Arginine) that projects from the alpha2 helix into the centre of the peptide-binding groove. While HLA-B*3501(+) individuals responded most strongly to the (123)IPSINVHHY(131) and (366)HPTFTSQY(373) epitopes, HLA-B*3508(+) individuals responded preferentially to (103)CPSQEPMSIYVY(114) and (188)FPTKDVAL(195). By comparing peptide-MHC association and disassociation rates with peptide immunogenicity, it was clear that dissociation rates correlate more closely with the hierarchy of immunodominance among the four pp65 peptides. These findings demonstrate that MHC micropolymorphism at positions outside the primary anchor residue binding pockets can have a major impact on determinant selection in antiviral T cell responses. Such influences may provide the evolutionary pressure that maintains closely related MHC molecules in diverse human populations.     

Identification of HLA-B44 and HLA-B45 alleles using a nested arms-PCR method

The establishment of DNA based identification of HLA Class I specificities has made high resolution typing possible at the allelic level. With such methods, it is feasible to identify population variation in allelic frequencies. Working towards higher allelic resolution of the HLA Class I loci we have developed a panel of primers to identify all the known HLA-B^*44 alleles and B^*4501 using a nested ARMS-PCR approach. There are currently seven different B^*44 alleles identified: B^*4402, B^*44031, B^*44032, B^*4404, B^*4405, B^*4406 and B^*4407. The system was validated using DNAs from B lymphoblastoid cell lines which have been sequenced for their B^*44 alleles. Further typing has been performed on a panel of 29 cell lines from the IHW cell panel and some 37 samples collected as part of the anthropological study of the Orkney islanders, all of which were previously defined as being B^*44 by the 12th Workshop HLA Class I typing kit. In the Orcadian population B^*4402 was found in 28 individuals to be the most common B^*44 allele, with 18 of these individuals expressing the A^*02-Cw^*0501-B^*4402 haplotype. The remaining 9 B^*44 positive individuals were all found to express the A^*29-Cw^*1601-B^*44031 haplotype. Methods and results will be presented.     

Analysis of endogenous peptides eluted from HLA-B27 variants: implications in the susceptibility to spondylarthropathies

Population studies suggest that some HLA-B27 subtypes (HLA-B^*2705, B^*2702) could be more strongly associated with the development of spondylarthropathies than others (B^*2703, B^*2706, B^*2709). Differences in the peptide binding groove could impose differences in the nature of peptides bound by these different alleles. We have eluted endogenous peptides from C1R-B^*2705 and B^*2703 transfectants. The B^*2705 HPLC profile was more complex than the B^*2703 one. Several B^*2705 and B^*2703 individual peaks were sequenced by Edman degradation and mass spectrometry. Some peptides were shared by both subtypes. One B^*2705 eluted peptide present in a major HPLC fraction was not found in the B^*2703 peptides. The corresponding synthetic peptide bound in vitro specifically to T2-B^*2705 and not to T2-B^*2703. This result emphasizes that even one amino-acid difference outside the major anchor binding pockets at position 59 between B^*2705 and B^*2703 could notably influence the endogenous peptides naturally presented. This could have consequences in terms of T cell repertoire selection and development of autoimmunity.     

B78Hen, a new Caucasian member within the B78 family

We have previously described an antigen B7801 (SNA) belonging into the B5 CREG, exclusively found in North and West African individuals. More recently, a new subtype B^*7802, differing from B^*7801 by two nucleotide substitutions within the class I heavy chain 1 domain has been found in a Caucasian individual (1).

Here we describe another B78 variant (B78Hen) that we observed in a Belgian German family. Serology displayed a pattern identical to the one of SNA. Careful analysis of the pedigree could rule out African ancestry. The exon 2 of B78Hen differed only by one nucleotide substitution from B^*7801 and B^*7802. In B78Hen, Asp at codon 74 of B^*7801 was substituted by Tyr, as in B^*7802. With respect to B^*7802, a silent mutation was observed in B78Hen at codon 23 (ATC to ATT). The ATT codon at that position is also found in most B51 subtypes, B^*52012 and B^*7801 and is absent in B^*51022, B^*5105, B^*7802 and B^*35.

Beside its phylogenetic implication, this finding has also practical consequences. The B78 antigens are not restricted to African subjects as it was previously thought. In view of its frequent confusion with B35 and B70 antigens, it must now be searched actively in Caucasian subjects.     

Peptide binding to the differentially disease-associated HLA-B2704 and B2706 and to mutants mimicking their polimorphism

Peptide binding to the disease-associated antigen HLA-B27 and its modulation by subtype polimorphism was addressed in this study. The effect of subtype changes was analyzed using a quantitative stabilization assay in which the surface expression of HLA-B27 on RMA-S cells was measured as a function of the concentration of peptides naturally presented by B^*2705, B^*2702 and their analogues. Binding to B^*2704, B^*2706 and to mutants mimicking the changes between these subtypes and B^*2705 was analyzed. Bulky aliphatic (Leu), aromatic (Phe or Tyr) or basic (Arg,, Lys) C-terminal residues contribute similarly to binding to B^*2705. For B^*2704 aliphatic C-terminal residues are the most suitable, but aromatic and even basic residues can also be accomodated. B^*2706 has strong preference for bulky aliphatic C-terminal residues and moderate suitability for aromatic ones. The effects of individual changes in the subtypes account only partially for the binding properties of B^*2704 and B^*2706, suggesting interactive effects of the changes in determining the peptide specificity of these subtypes that are not fully accounted for by simple additive effects of the individual changes.