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.     

B*2707 differs in peptide specificity from B*2705 and B*2704 as much as from HLA-B27 subtypes not associated to spondyloarthritis

HLA-B*2707 is associated with ankylosing spondylitis in most populations. Like the non-associated allotypes B*2706 and B*2709, it lacks Asp116 and shows preference for peptides with nonpolar C-terminal residues. The relationships between the peptide specificity of B*2707 and those of the disease-associated B*2705 and the non-associated subtypes were analyzed by determining the overlap between the corresponding peptide repertoires, the sequence of shared and differential ligands, and by comparing allospecific T cell epitopes with peptide sharing. The B*2707-bound repertoire was as different from that of B*2705 as from those of B*2706, B*2709, or the two latter subtypes from each other. Differences between B*2707 and B*2705 were based on their C-terminal residue specificity and a subtle modulation at other positions. Differential usage of secondary anchor residues explained the disparity between the B*2707-, B*2706-, and B*2709-bound repertoires. Similar differences in residue usage were found between B*2707 and both B*2704 and B*2706, as expected from the high peptide overlap between the two latter subtypes. T cell cross-reaction paralleled peptide sharing, suggesting that many shared ligands conserve their alloantigenic features on distinct subtypes. Our results indicate that association of HLA-B27 subtypes with ankylosing spondylitis does not correlate with higher peptide sharing among disease-associated subtypes or with obvious peptide motifs.     

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.     

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.     

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-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.     

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.     

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 (B*2701) specificity for peptides lacking Arg2 is determined by polymorphism outside the B pocket

B*2701 differs from B*2705 by three amino acid changes: DY74, DN77, LA81, and from B*2702 only by two: DY74 and T180. Tyr74 is located in the C/F cavity of the peptide-binding site, and is unique to B*2701 among HLA-B27 subtypes. Binding of natural B*2705 and B*2702 ligands to B*2701, and to mutants mimicking subtype changes, was analyzed. In addition, sequencing of the peptides bound in vivo by B*2701 and the Y74 mutant was carried out. The main distinctive feature of B*2701 was its presentation of peptides with Gln2. Synthetic analogs bound in vitro similarly as the corresponding ligands with Arg2. Moreover, both Gln2 and Arg2 were dominant upon pool sequencing of B*2701- bound peptides, and 2 of 8 natural ligands contained Gln2. Suitability of Gln2 was largely determined by the Y74 change, as indicated by: 1) binding of Gln2 analogs to this mutant, and 2) detection of Gln2 by pool sequencing of Y74-bound peptides. B*2701 bound peptides with C-terminal aromatic or Leu residues, and interacted with these motifs more strongly than B*2702. The Y74 mutation alone was not responsible for poor binding of peptides with C-terminal basic residues to B*2701, since they bound efficiently and at least one was presented in vivo by this mutant. Most peptides bound to the A81 mutant worse than to B*2705, but frequently better than to B*2701 or B*2702, suggesting that other subtype changes were compensatory. The peptide specificity of B*2701 suggests that this subtype may determine susceptibility to spondyloarthropathy.     

The South Amerindian allotype HLA-B*3909 has the largest known similarity in peptide specificity and common natural ligands with HLA-B27

HLA-B*3909 has only been found among South Amerindians, and presumably arose locally in these populations. It differs from B*3901 by a single Tyr to Ser change at position 99. To analyze the influence of this polymorphism on peptide specificity, pool sequence analysis and sequencing of multiple individual ligands from B*3901 and B*3909 were carried out. Both allotypes bind peptides with Arg2 or His2 and nonpolar C-terminal residues. However, whereas His2 is the predominant B*3901 motif, a majority of the B*3909-bound peptides have Arg2. In addition, B*3909 binds peptides with Pro2, and also shows an increased preference for Pro3. In spite of their differences, both subtypes bind overlapping peptide repertoires, as indicated by the identification of several identical ligands from their respective peptide pools. B*3909 is significantly more similar in its peptide specificity to HLA-B27 than B*3901. This is due to the increased preference of B*3909 for Arg2 and to low suitability of HLA-B27 for His2. The similarity between HLA-B27 and B*3909 was confirmed by identification of a natural ligand common to both allotypes. In addition, multiple HLA-B27 ligands bound efficiently B*3909 in vitro. The results indicate that, among the HLA class I allotypes of known peptide specificity, B*3909 is the most similar in its peptide binding properties to HLA-B27, which is absent in South Amerindians. This may have implications for the susceptibility of individuals in these populations to spondyloarthropathies.     

Structure of HLA-B27-specific T cell epitopes. Antigen presentation in B2703 is limited mostly to a subset of the antigenic determinants on B2705

The structure of HLA-B27-specific epitopes recognized by anti-B*2705 and anti-B*2703 cytotoxic T lymphocytes (CTL) from three unrelated donors was examined with site-specific mutants at various side-chain pockets in the antigen-binding site. The effect of any given mutation on allorecognition correlated strongly with its predictable effect on peptide binding. Acidic charges in the C/F pocket of HLA-B27, which binds C-terminal peptide residues, strongly modulated allorecognition. Anti-B*2705 CTL from different donors were differently affected by some mutations, indicating individual differences in the structure of epitopes recognized by alloreactive CTL from each donor. Most anti-B*2703 CTL recognized a subset of epitopes that were also present on B*2705, but differed from the bulk of allospecific epitopes on this subtype in their smaller dependence on pocket A structure, where the difference between these subtypes is located, and in their greater dependence on Glu45, in the B pocket. The structure of the very few epitopes on B*2703 not shared by B*2705 was quite different from that of the much more predominant cross-reactive epitopes. The results strongly suggest that B*2703 is antigenically defective as compared with B*2705 and that this is due to the fact that the repertoire of peptides presented by B*2703 consists mainly of a subset of the B*2705-bound peptides which do not critically require the canonic binding of the peptidic N-terminus to a B*2705-like A pocket, because they are sufficiently stabilized by other contacts through the peptide binding site.     

A single amino acid change makes the peptide specificity of B*3910 unrelated to B*3901 and closer to a group of HLA-B proteins including the malaria-protecting allotype HLA-B53

Abstract: HLA-B*3910, which has only been found in African and African American individuals, differs from B*3901 by the single amino acid change of Cys67 to Tyr67. Sequence analysis of the B*3910-bound peptide pool and of several individual ligands revealed that this subtype has strong preference for peptides with Pro2. This is in contrast with the preference of B*3901 for peptides with basic residues (Arg and His) at this position, and indicates that the single amino acid substitution between B*3910 and B*3901 totally changes the repertoire of bound peptides. This is presumably due to the significant decrease in the size of the B pocket, and to its increased hydrophobicity, since Tyr67 takes part in this pocket. B*3910 is similar to various other class I proteins in its preference for peptides with Pro2 and nonpolar C-terminal residues, including HLA-B53, an antigen associated with protection against severe malaria. The role of these two motifs as major peptidic anchors suggests that B*3910 and HLA-B53 may bind common peptides.     

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-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%).     

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.     

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.     

Modulation on immunogenicity by HLA-B27 subtype polymorphism

Cells from the same HLA-B27- individual, PA, were stimulated in vitro in primary mixed lymphocyte culture, with either B*2705+ or B*2704+ lymphoblastoid cell lines, in independent experiments. Cytolytic T lymphocytes (CTL) were cloned at limiting dilution and the clones obtained were screened for anti-B27 alloreactivity. Most of the CTL clones generated against the B*2705+ stimulator cells were directed against the B*2705 antigen. In contrast, no anti-B27 CTL clones were found among those derived against the B*2704+ stimulator cells. This was not due to a poor cytotoxic response against these cells because a large proportion of the T cell clones derived from this stimulation were cytotoxic. B2704 differs from B*2705 by only two amino acid changes at positions 77 and 152. Previous studies (Aparacio, P. et al., Eur. J. Immunol. 1988.18: 203) have shown that none of the anti-B*2705 CTL clones derived from donor PA and amenable to detailed characterization cross-reacted with B*2704, suggesting that most of this cytotoxic response was directed against an immunodominant determinant contributed for by residues 77 and/or 152 from B*2705. The present results further suggest that the changes at these positions in B*2704 alter this determinant in such a way that B*2704 becomes less immunogenic for the particular individual PA. Furthermore, a similar poor anti-B*2704 CTL response was obtained from a second B27- responder individual, AE, stimulated with another B2704+ cell line. The single anti-B*2704 CTL clone, 64.8P, isolated from this second individual, displayed an unusual reaction pattern in that it cross-reacted with all B27 subtypes with changes only at or close to positions 77 and 152, including B*2705. Significantly, the only HLA-B27 subtype that was not recognized by CTL 64.8P was B*2703, which differs from B*2705 only at residue 59. This residue is located in the three-dimensional structure at the opposite end from residues 77 and 152 at the surface of the antigen-binding groove of the class I molecule. Thus, the area around residues 77 and 152 is not an essential part of the epitope recognized by CTL 64.8P.     

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.     

Identification of a novel HLA-B*2722 allele from a Filipino cell

We present the novel HLA-allele B*2722 amplified and sequenced from a Filipino individual. This DNA was included several times in the International Cell Exchange, UCLA, and typed at the time as B*27 or B*2706 by the majority of the participating laboratories. The second HLA-B allele of this person is B*3802. B*2704/06 or B*2704/06/10 were suggested by approximately one-third of the laboratories. As we were investigating the intron 3 sequences of B*27 alleles, we also sequenced exon 4 of this Filipino DNA and found a single nucleotide exchange in exon 4 (pos. 704) which was not in concordance with the previous B*2706 typing. Our sequencing results showed that the coding sequence of B*2722 is identical to B*2706 in exon 1, 2 and 3. In exon 4 the B*2722 sequence is identical to B*2704. HLA-B*2704 differs from B*2706 in one base at position 704, a G in B*2704 and a C in B*2706, respectively.