Lack of carboxyl–terminal tyrosine distinguishes the B2706–bound peptide repertoire from those of B2704 and other HLA–B27 subtypes associated with ankylosing spondylitis

B*2704 and B*2706 are two closely related HLA-B27 subtypes, which differ from the common B*2705 by the Asp>Ser77, Val>Glu152, and Ala>Gly211 amino acid changes. In addition, B*2706 differs from B*2704 by the His>Asp114 and Asp>Tyrl 16 changes. In spite of their similarity B*2704, but not B*2706, was associated to ankylosing spondylitis in a same population. We have carried out pool sequence analyses of the peptides naturally bound to each of these subtypes, and of several individual peptide ligands. B*2704 and B*2706 shared with B*2705, among other features, their selectivity for Arg² and their allowance for some aliphatic and aromatic C-terminal residues in their bound peptides. The main features that distinguished both subtypes from B*2705 were: 1) their failure to present peptides with C-terminal basic residues, and 2) their allowance for both polar and nonpolar residues at peptide position 3. A major difference between B*2704 and B*2706 was that C-terminal Tyr was prominent among the peptides bound to B*2704, but was not detected among those from B*2706. The use of Tyr as a C-terminal anchor motif is the only functional feature shared by the disease-associated B*2705, B*2702, and B*2704 subtypes that is absent in B*2706. This suggests that the ability of HLA-B27 to present peptides with C-terminal Tyr might be critical for its association to spondyloarthropathy,     

Large sharing of T-cell epitopes and natural ligands between HLA-B27 subtypes (B*2702 and B*2705) associated with spondyloarthritis

HLA-B*2702 is an ankylosing spondylitis-associated allotype that differs from the more common B*2705 at residues 77, 80, and 81, in the peptide-binding site. The diversity and fine specificity of alloreactive cytolytic T-lymphocyte (CTL) raised against B*2702 were analyzed at the clonal level. Significant crossreaction with B*2705 and B*2709 indicated that the three subtypes share numerous T-cell epitopes. However, some epitopes shared by B*2702 and B*2705 were lost in B*2709, which correlates with weaker association of this subtype to disease. Clonal specificities were donor-dependent, indicating that allo-immunogenicity is variable among individuals. Anti-B*2702 CTL were little affected by single mutations mimicking B*2702/B*2705 polymorphism, but the double mutant at positions 77 and 81 was recognized worse than B*2705, suggesting a compensatory effect of residue 80. Thus, HLA-B27 polymorphism modulated alloreactivity through cooperative and compensatory effects on T-cell epitope structure. Comparison of B*2705- and B*2702-bound peptide repertoires revealed that they overlapped by 73% and 81%, respectively. This was larger than B*2702/B*2705 cross-reaction, indicating that HLA-B27 allospecificity is only partially determined by the nature of peptide repertoires. The large sharing of natural ligands and T-cell epitopes is consistent with a pathogenetic role of B*2702 and B*2705 in spondyloarthritis based on antigen presentation.     

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.     

HLA-B27 presents a peptide from a polymorphic region of its own molecule with homology to proteins from arthritogenic bacteria

A possible mechanism for the pathogenesis of HLA-B27-associated spondyloarthropathies is that peptides from arthritogenic bacteria with homology to endogenous self-peptides presented by HLA-B27, including those derived from HLA-B27 itself, could elicit an autoimmune T-cell response upon infection. We report here that an undecamer corresponding to the polymorphic region of HLA-B27 spanning residues 169–179 is presented in vivo by the B*2701, B*2704 and B*2706 subtypes, but was not detected in the B*2703-bound peptide pool. This peptide binds to B*2705 in vitro with sufficient affinity to allow its natural presentation by this subtype, but it binds with low affinity to B*2703. In spite of homology of this peptide to proteins from arthritogenic bacteria, its binding specificity does not correlate with current evidence concerning association of HLA-B27 subtypes to ankylosing spondylitis, suggesting that presentation of this peptide is not the critical feature that determines linkage of HLA-B27 to this disease.     

Discrimination of HLA-B27 alleles by group-specific amplification followed by solid-phase sequencing

HLA-B27 is known to be highly associated with ankylosing spondylitis. Until now, nine B27 subtypes have been sequenced and may contribute in different fashions to ankylosing spondylitis. Additionally, the divergent subtypes may be of clinical importance in bone marrow transplantation with alternative donors. The purpose of this study was to determine the different subtypes of HLA-B27 by a direct sequencing approach. The typing strategy is based on a group-specific amplification of the second and third exon followed by automated fluorescence sequencing of the polymorphic regions. The extensive sharing of sequence motifs between the different B alleles made it impossible to specifically amplify the B27 group under the precondition of including all sequence variations necessary for a postamplification specificity step. Therefore, for setting up a direct sequencing approach of B27, co-amplified B alleles had to be taken into account. In order to get unambiguous sequencing chromatograms without any heterozygous positions, nested sequencing primers were used which selectively matched sequence motifs only present in the second and third exon of the amplified B27 alleles. This strategy allowed in all cases investigated a clear separation of the haplotypes, revealing unequivocal sequencing results. Using this method, we have investigated 93 B27-positive individuals. Sequencing identified the alleles B*2702, 2703, 2704, 2705, and 2707. B*2701, 2706, 2708, and 2709 were not represented in the population studied.     

Diversity of human leukocyte antigen-B27 alleles in Han population of Hunan province, southern China

This study was to investigate the frequency of HLA-B27 and its subtypes in the Han population of Hunan province, southern China. One hundred and sixty-nine healthy unrelated donors were tested for HLA-B27 by polymerase chain reaction-sequence-specific primer (PCR-SSP). One hundred and twenty-eight B27-positive spondyloarthropathy patients and 18 B27-positive healthy controls were subtyped using the high-resolution PCR-SSP. The phenotype frequency of human leukocyte antigen (HLA)-B27 was found to be 2.36% in healthy population. Five B27 alleles were identified: B*2704, B*2705, B*2706, B*2707, and B*2724. No significant difference was found in the distribution of HLA-B27 subtypes between the patients and controls studied. Notably, B*2724 was observed in a juvenile patient with ankylosing spondylitis. This subtype has not been previously reported in Chinese ankylosing spondylitis (AS) patients and other ethnic groups.     

Binding of peptides naturally presented by HLA–B27 to the differentially disease–associated B*2704 and B*2706 subtypes, and to mutants mimicking their polymorphism

B*2704 and B*2706 are closely related HLA–B27 subtypes of which the former but not the latter is associated to ankylosing spondylitis. Their peptide specificity relative to other disease–associated subtypes was analyzed by testing binding of self–peptides naturally presented by B*2705 or B*2702, and synthetic analogs, to B*2704, B*2706, and site–specific mutants mimicking their changes. Peptides with basic, aliphatic or aromatic C–terminal residues bound to B*2705 with similar affinity. In B*2704 C–terminal aliphatic/aromatic residues were preferred. B*2706 discriminated drastically between polar and nonpolar C–terminal residues, showing strong preference for Leu and Phe, and less than B*2704 for basic and Tyr residues. Loss of single acidic charges (D>S77, D>Y116) increased preference for C–terminal Leu and Phe, but allowed efficient binding of peptides with basic residues or Tyr. Their gain (V>E152, H>D114) maintained wide C–terminal specificity, but severely impaired binding, presumably by disrupting interactions with internal peptide residues. This was compensated by Y116 in the double Dl 14Y116 mutant. The specificity of B*2704 and B*2706 was explained only partially by the separate effects of single mutations, indicating that novel properties arise from concomitant changes at various positions. For instance, specificity of B*2706 for nonpolar C-terminal residues required simultaneous removal of Asp⁷⁷ and Asp116. B*2706 differed from B*2705, B*2702, and B*2704 in its lower suitability for C-terminal Tyr, suggesting that this feature might be relevant for HLA–B27 association to spondyloarthropathy.     

The naturally occurring polymorphism Asp116 → His116 , differentiating the ankylosing spondylitis-associated HLA-B*2705 from the non-associated HLA-B*2709 subtype,influences peptide-specific CD8 T cell recognition

HLA-B27 molecules are interesting because of their strong association with ankylosing spondylitis (AS) and reactive arthritis (ReA). A pathogenetic role for these molecules has been postulated in presenting a putative --bb--arthritogenic peptide to CD8 T cells. The HLA-B*2709 subtype, although differing by a single amino acid (His¹¹⁶ → Asp¹¹⁶ ) from the wide spread and strongly AS-associated subtype HLA-B*2705, is not found in patients. Since residue 116 interacts with the C terminus of the peptide, it is possible that the two subtypes differ in their antigen-presenting features. We show here that CD8 T cells can distinguish the two HLA-B27 subtypes when presenting a same epitope derived from Epstein-Barr virus-latent membrane protein 2. Moreover, alanine scanning mutagenesis analysis revealed that the peptide residues relevant for such recognition are different depending on whether HLA-B*2705 or -B*2709 molecules present the epitope. These results give support to the belief that functional differences determined by subtype-specific polymorphisms can have a pathogenetic relevance and open up a new scenario where subtle modifications within the peptide/HLA ligand might be responsible for the differential association between HLA-B27 subtypes and spondyloarthropathies.     

少儿强直性脊柱炎与幼儿类风湿性关节炎HLA-B27亚型的鉴别诊断

目的：探讨HLA－B27等位基因亚型与少年强直性脊柱炎和幼年类风湿性关节炎的关联。方法：用PCR－SSP方法对74人HLA－B27等位基因亚型进行研究，其中少年强直性脊柱炎32例，幼年类风湿性关节炎28例，5个家系中患者的父亲或母亲5例，正常对照组9例，并进行关联分析。结果：本组人群的HLA－B27等位基因由HLA－B*2704、*2705、*2702、*2707 4种亚型组成，其中少年强直性脊柱炎患者HLA－B27等位基因亚型频率为B*2704 56.25%、B*2705 40.63%、B*2702 3.13%；幼年类风湿性关节炎HLA－B27等位基因亚型频率为B*2705 60．7％、B*2704 28．57％、B*2702 3．57％及B*2707为7．14％；少年强直性脊柱炎与幼年类风湿性关节炎结果比较，HLA－B*2704基因频率在少年强直性脊柱炎组高于幼年类风湿性关节炎组（RR＝3．21，P＜0．05）。结论：少年强直性脊柱炎与HLA－B*2704等位基因亚型关联。对HLA－B27等位基因亚型的检测可成为少年强直性脊柱炎和幼年类风湿性关节炎鉴别诊断中一个有价值的实验指标。     

HLA-B*27 subtypes in Northern and Northeastern Thais, Karens, and Bamars determined by a high-resolution PCR-SSP technique

Human leukocyte antigens (HLA), class I, are a group of antigens expressed on most nucleated cell surfaces. They transport endogenous peptides to the cell surface for recognition by T-cell receptors. Their functions are involved in immune responses. Many diseases are associated with HLA alleles, especially HLA-B*27 that is strongly associated with ankylosing spondylitis (AS). HLA-B*27 consists of 42 subtypes. Different subtypes of HLA-B*27 were reported in different ethnic groups of AS patients. In this study, a high-resolution polymerase chain reaction–sequence-specific primer technique has been developed to define all the HLA-B*27 subtypes with a total of 29 primer mixtures. Two of the primer mixes were used to detect the HLA-B*27 -specific group, and 27 primer mixes were used to identify 42 subtypes ( B*2701–B*2721 and B*2723–B*27 43). The HLA-B*27 -group-specific primers have been tested in unrelated healthy subjects; 846 Northeastern Thais (NET), 334 Northern Thais (NT), 264 Karens, and 310 Bamars. Sixty-three NET (phenotype frequency, PF = 7.4%), 24 NT (PF = 7.1%), 5 Karens (PF = 1.8%), and 12 Bamars (PF = 3.9%) were positive for HLA-B*27 . Only B*2704 was found in Karens, whereas B*2704 , B*2705/37/39 , B*2706 , and B*2707 were found in NET and NT. In Bamars, B*2704 , B*2705/37/39 , B*2706 , and B*2725 were found. The distribution of HLA-B*27 subtypes was compared with other studies in Asian and Caucasian populations. Significant differences of the distribution of HLA-B*27 subtypes were found in most of the populations. This study established a simple technology for HLA-B*27 subtyping and provided basic information for anthropology and further studies in disease associations.     

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.     

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.     

HLA-B27 polymorphism and worldwide susceptibility to ankylosing spondylitis

HLA-B27 is strongly associated to ankylosing spondylitis (AS) and represents a family of eleven B27 alleles (B*2701–11). Our aim was to analyze the distribution of B27 subtypes by PCR/SSOP and genomic sequencing in a large group of populations ( n =17). 711 B27-positive samples from Caucasoid, Asian, African, Amerindian and Polynesian populations were selected to ascertain transracial gene mapping of the B27 subtypes. 476 of these were AS patients, chosen to investigate the contribution of B27 alleles to AS susceptibility. Some significant new findings have arisen from this study: 1) B*2705 was the predominant subtype in circumpolar and subarctic areas. B*2702 was found to be practically restricted to Caucasian populations, showing a higher frequency in Middle-East (Jews) and North Africa (Arabs/Berbers) groups. 2) B*2703 appears associated with AS in Western Africans. This is of remarkable interest since it was suggested that B*2703 would be negatively disease-associated. 3) Although B*2706 appears negatively associated with AS in Thais, we identified two patients from northern China carrying it. This may be a reflection of a disease heterogeneity and could indicate that more than one pathogenic agent can be involved in AS. B*2709 has been recently described as negatively associated with AS in Sardinians. The molecular changes His 114Asp (B*2706) and Asp 116His (B*2709) could modify the genetic susceptibility to AS.     

HLA-B27 in the Greek Cypriot population: distribution of subtypes in patients with ankylosing spondylitis and other HLA-B27-related diseases. The possible protective role of B*2707

The major purpose of the present study was to investigate the frequency of human leukocyte antigen (HLA)-B27 alleles in healthy controls and in patients with ankylosing spondylitis (AS) and other HLA-B27–related diseases in the Greek Cypriot population. We selected 102 HLA-B27–positive individuals (60 controls and 42 patients). Typing of the HLA-B27 alleles was performed by polymerase chain reaction amplification with sequence-specific primers. Only two alleles were detected in the patient group: B*2702 (n = 31, 73.8%) and B*2705 (n = 11, 26.2%). The HLA-B*2707 allele was detected (n = 10, 16.7%) only in the healthy controls in addition to the B*2702 (n = 31, 51.7%) and B*2705 (n = 19, 31.7%) alleles. Our results show a restricted number of HLA-B27 subtypes associated with AS and other B27-related diseases and an elevated frequency of the B*2702 allele in the AS patients. The allele B*2707 seems to have a protective role in the population studied because it was found only in the healthy controls.     

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.     

New insights regarding HLA-B27 diversity in the Asian population

A polymerase chain reaction-sequence-specific primer (PCR-SSP) method which distinguishes all B27 alleles described at present (B*2701-23) has been developed. The distribution of B27 alleles was characterised in six different Asian populations. HLA-B*2705, 02, 04, 07, 22 (formerly B*2706) subtypes found in Asian populations differ in their ethnic distribution, which may be the result of different genetic and geographic origins. Furthermore, two novel B27 alleles were found in this study. B*2714 was identified in two Siberians, one of whom was a patient with ankylosing spondylitis. B*2715 was found in two patients with ankylosing spondylitis in Thais. These associations have not previously been reported in either ethnic group.     

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.     

Frequency of HLA-B27 subtypes in a Danish population and in Danish patients with ankylosing spondylitis

Polymerase chain reaction in combination with sequence-specific oligonucleotide probes were used to analyze nine HLA-B27 subtypes among 51 healthy I HLA-B27 positive Danish blood donors and 30 Danish HLA-B27 positive patients with ankylosing spondylitis (AS). In the group of healthy Danes we found two subtypes, B*2705 (90.2%) and B*2702 (9.8%), however, among the AS patients only the B*2705 subtype was detected. We did not find a significant evidence for associations between AS and a particular HLA-B27 subtype in a Danish population.     

HLA-B27 and ankylosing spondylitis: tales from China

Abstract
The two most frequent HLA-B27 subtypes worldwide are B*2704 and B*2705. In the Han population of China B*2704 and, to a lower extent, B*2705 are found with significant frequency, and both are associated to ankylosing spondylitis (AS). Two articles in this issue report that the association to AS in this ethnic group is stronger for B*2704 than for B*2705. Thus, at least among the Han, B*2704 would be the strongest known susceptibility factor for AS.     

HLA-B27 polymorphism in the Malays

The frequency of HLA-B27 and its subtypes was determined in 878 Malay subjects. Thirty-five of the subjects typed for HLA-A, -B and -DR were found to be positive for HLA-B27. The frequency of this allele in the Malay population was found to be 3.99%. The subtypes observed and their frequencies are: HLA-B*2704 (19.4%), HLA-B*2705 (5.6%), HLA-B*2706 (72.2%) and HLA-B*2707 (2.8%).