用组合肽库技术分析HLA-B27的抗原提呈模式

HLA分子抗原表位提呈模式的分析,在自身免疫病和肿瘤的病因与治疗研究方面有重要意义。本研究采用组合肽库的策略合成19组ORX7型肽亚库,通过与荧光素标记肽的竞争结合试验,分析了与强直性脊柱炎有强相关的HLA-B27分子的抗原提呈模式。结果显示HLA-B27与P1为不同氨基酸残基的19种肽亚库有相近的结合率,提示P1为非锚定残基;中国人群最常见的二种HLA-B27亚型B*2704和B*2705,在提呈肽表位的P1模式方面存在一些小差异,P1为D或E的肽亚库与HLA-B*2704的结合能力要强一些,而P1为K的肽亚库则与HLA-B*2705的结合能力强一些。本研究为HLA-B27与强直性脊柱炎关联机制的研究提供了线索,为开展HLA分子的抗原提呈模式分析打下了基础。     

Crosses of two independently derived transgenic mice demonstrate functional complementation of the genes encoding heavy (HLA-B27) and light (beta 2-microglobulin) chains of HLA class I antigens

In man a number of diseases are associated with certain alleles of MHC antigens. The most pronounced example is ankylosing spondylitis, which is strongly associated with HLA-B27. As a first step towards a model system to study the basis of this association, transgenic mice were generated that showed cell surface expression of the HLA-B27 antigen biochemically indistinguishable from HLA-B27 antigen expressed on human cells. This result was obtained by crossing two independently derived strains of mice, one of which is transgenic for the HLA-B27 heavy chain gene, and the other carrying and expressing the human beta 2m gene. Examination of HLA-B27 and human beta 2m mRNA in various tissues shows the two genes to be expressed in a coordinate fashion. The mRNA levels follow those of endogenous H-2 Class I genes.     

Natural MHC class I polymorphism controls the pathway of peptide dissociation from HLA-B27 complexes

Analysis of antigen dissociation provides insight into peptide presentation modes of folded human leukocyte antigen (HLA) molecules, which consist of a heavy chain, beta2-microglobulin (beta2m), and an antigenic peptide. Here we have monitored peptide-HLA interactions and peptide dissociation kinetics of two HLA-B27 subtypes by fluorescence depolarization techniques. A single natural amino-acid substitution distinguishes the HLA-B*2705 subtype that is associated with the autoimmune disease ankylosing spondylitis from the non-disease-associated HLA-B*2709 subtype. Peptides with C-terminal Arg or Lys represent 27% of the natural B*2705 ligands. Our results show that dissociation of a model peptide with a C-terminal Lys (GRFAAAIAK) follows a two-step mechanism. Final peptide release occurs in the second step for both HLA-B27 subtypes. However, thermodynamics and kinetics of peptide-HLA interactions reveal different molecular mechanisms underlying the first step, as indicated by different activation energies of 95+/-8 kJ/mol (HLA-B*2705) and 150+/-10 kJ/mol (HLA-B*2709). In HLA-B*2709, partial peptide dissociation probably precedes fast final peptide release, while in HLA-B*2705 an allosteric mechanism based on long-range interactions between beta2m and the peptide binding groove controls the first step. The resulting peptide presentation mode lasts for days at physiological temperature, and determines the peptide-HLA-B*2705 conformation, which is recognized by cellular ligands such as T-cell receptors.     

A single residue exchange between two HLA-B27 alleles triggers increased peptide flexibility

For more than 30 years, human leukocyte antigen B27 (HLA-B27) has been known to be closely related to the autoimmune disease ankylosing spondylitis, yet little is known about the molecular mechanisms of pathogenesis. Crystal structures of two closely related, but differently disease-associated, subtypes (B*2705 and B*2709) also did not resolve this situation as they revealed the bound nonapeptide in essentially identical conformations. As the peptide is part of putative binding epitopes for the T cell receptor, we performed molecular dynamics simulations to gain deeper insight into the dynamic behaviour of HLA-B27 molecules. We find increased flexibility of the peptide in the binding groove of subtype B*2709 due to weaker interactions in the F pocket. Possible implications of this flexibility for T cell recognition and signalling are discussed.Abbreviations ₂m ₂-microglobulin - AS ankylosing spondylitis - CDR complementarity determining region - HC heavy chain - HLA human leukocyte antigen - MD molecular dynamics - MHC major histocompatibility complex - pMHC peptide-loaded MHC - RMSD root mean square deviation - RMSF root mean square fluctuation - TCR T cell receptor An erratum to this article can be found at     

HLA-B27 subtypes differentially associated with disease exhibit conformational differences in solution

Human leukocyte antigen (HLA) class I molecules consist of a heavy chain, β₂-microglobulin, and a peptide that are noncovalently bound. Certain HLA-B27 subtypes are associated with ankylosing spondylitis (such as HLA-B*2705), whereas others (such as HLA-B*2709) are not. Both differ in only one residue (Asp116 and His116, respectively) in the F pocket that accommodates the peptide C-terminus. An isotope-edited IR spectroscopy study of these HLA-B27 subtypes complexed with the self-peptide RRKWRRWHL was carried out, revealing that the heavy chain is more flexible in the HLA-B*2705 than in the HLA-B*2709 subtype. In agreement with these experimental data, molecular dynamics simulations showed an increased flexibility of the HLA-B*2705 binding groove in comparison with that of the HLA-B*2709 subtype. This difference correlates with an opening of the HLA-B*2705 binding groove, accompanied by a partial detachment of the C-terminal peptide anchor. These combined results demonstrate how the deeply embedded polymorphic heavy-chain residue 116 influences the flexibility of the peptide binding groove in a subtype-dependent manner, a feature that could also influence the recognition of the HLA-B27 complexes by effector cells.     

Systematic Identification and Comparative Analysis of Human Cartilage-Derived Self-peptides Presented Differently by Ankylosing Spondylitis (AS)-Associated HLA-B*27:05 and Non-AS-associated HLA-B*27:09

International Journal of Peptide Research and Therapeutics - The human leukocyte antigen B27 (HLA-B27) gene has been observed to significantly increase the risk of developing ankylosing spondylitis...     

The immunobiology of HLA-B27: variations on a theme

In the thirty years since the initial discovery of a striking association between HLA-B27 and susceptibility to ankylosing spondylitis, numerous hypotheses have been proposed to explain the role of this molecule in the pathogenesis of spondyloarthropathies. In the past few years the focus has shifted from one centered largely on the physiological peptide-presenting function of HLA-B27, to include ideas based on aberrant aspects of its immunobiology. This has been driven in part by results from animal models of HLA-B27-associated disease where CD8+ T cells do not appear to be playing a major role in pathogenesis. In addition, the HLA-B27 heavy chain is unusual in that it has a tendency to misfold in the endoplasmic reticulum and to form disulfide linked heavy chain dimers that can be expressed on the cell surface. Although the data suggest misfolding and cell surface dimerization are fundamentally different processes, it appears that certain structural features of the heavy chain are common to both. Potential links between these aberrant characteristics of HLA-B27 and inflammatory disease are discussed in this and other reviews in this issue. Herein we consider how protein misfolding affects cell function through the activation of an 'unfolded protein response' and/or an 'ER overload response', and the potential impact on the immune system. Despite significant advances in the treatment of spondyloarthropathies over the past few years, a better understanding of pathogenesis is likely to improve outcome by identifying ways to provide greater and more sustained clinical responses.     

HLA-B27分子及其与强直性脊柱炎关联的研究进展简

HLA-B27是MHCI类分子,因而具有MHCI类分子的相关生物学特性。在正常免疫状态下,HLA-B27分子执行对内源性抗原如肿瘤及病毒的免疫监视及杀伤功能;但在机体免疫状态出现异常的情况下,它又可能成为引发自身免疫性疾病的主要相关因素,其中最为著名的是其与强制性脊柱炎（As）的关联研究。我们首先概述了HLA—B27分子正常免疫学功能的研究进展,然后对其在AS相关性研究中的进展进行了总结和分析。通过对上述两方面的综述与分析,全方位展示HLA-B27在执行免疫学功能和引发免疫病理性改变方面的作用,为全面认识和深入研究HLA-B27分子的生理与病理学作用提供坚实的基础。     

Genetic susceptibility to ankylosing spondylitis

Ankylosing spondylitis is a highly heritable, common rheumatic condition, primarily affecting the axial skeleton. The association with HLA-B27 has been demonstrated worldwide, and evidence for a role of HLA-B27 in disease comes from linkage and association studies in humans, and transgenic animal models. However, twin studies indicate that HLA-B27 contributes only 16% of the total genetic risk for disease. Furthermore, there is compelling evidence that non-B27 genes, both within and outwith the major histocompatability complex, are involved in disease aetiology. In this post-genomic era we have the tools to help elicit the genetic basis of disease. This review describes methods for genetic investigation of ankylosing spondylitis, and summarises the status of current research in this exciting area.     

Interesting relations in the HLA system

There exists no absolute binding between the antigens HLA-Cw 2, Cw 3 and Cw 4, on the one hand, and HLA-B 27, HLA-B 15 and HLA-Bw 35, on the other hand. Even if 91% of human beings with HLA Cw 4 will simultaneously have the antigen HLA-Bw 35, another antigen as HLA-B 27 or HLA-B 15 can be identified in approximately 55% of individuals with HLA-Cw 2 and Cw 3. In this connection, the joint presence of some pairs of cross-reacting HLA antigens (A 2 and A 28, B 5 and Bw 35, B 7 and B 27, B 8 and B 14, B 12 and Bw 2) could be proved and their frequency be determined. 2 cases of a simultaneous presence of two subtypes of HLA-A 10 antigen, A 25 and A 26, could be found in family examinations. Moreover, two atypical bindings of anti-HLA-Bw 4 and anti-HLA-Bw 6 cytotoxins with HLA antigens could be identified: 7,49% of HLA-Bw 35 positive lymphocytes no positive response with anti-HLA-B 4 and 1,69% of HLA-B 12 with anti-HLA Bw 6. The importance of the findings for determining HLA in practice is discussed.     

The relationship between genetics and environment in the pathogenesis of rheumatic diseases

Major developments have taken place to further our understanding of the relationship between genetics and the environment in the pathogenesis of rheumatic disorders. The association between HLA markers and human disease is becoming clearer. For instance, HLA-DRW4 frequently occurs in patients with rheumatoid disease, and penicillamine and gold toxicity are seen most often in patients with HLA-DRW2 or DRW3. Antisera to B alloantigens help define the genetic differences between systemic lupus erythematosus and rheumatoid arthritis. As yet, the most dramatic link is that between HLA-B27 and primary ankylosing spondylitis. This same antigen is related, to varying degrees, with other members of the seronegative spondylarthritides and there is strong evidence that this association is related to HLA-B27, itself, rather than an associated disease gene. Nevertheless, some data refute a single gene theory. We are just beginning to learn more about interactions between different genes on the sixth chromosome and genes on other chromosomes.The sex ratio of the spondylarthritides is now better defined. Sacroiliitis may have a comparable sex distribution although females have more peripheral joint disease and males have greater spinal involvement. Unfortunately, the explanation for these differences remains elusive.The specific infective agents related to the development of rheumatic disorders are becoming clarified. Chlamydia, Salmonella, Yersinia and Shigella flexneri types 1b and 2a are arthritogenic, while Shigella sonnei appears not to cause disease. Although the Reiter syndrome is now considered a chronic disease, the reason for remissions and relapses remains unclear.     

Citrullination-dependent differential presentation of a self-peptide by HLA-B27 subtypes

Inflammatory processes are accompanied by the posttranslational modification of certain arginine residues within proteins to yield citrulline, although it is largely unknown how this modification influences antigen presentation. We employed crystallographic and functional studies to investigate whether the exchange of arginine to citrulline affects the display of a peptide by two human major histocompatibility antigen class I subtypes, HLA-B(*)2705 and HLA-B(*)2709. Both differ only in residue 116 within the peptide binding groove despite their differential association with ankylosing spondylitis, an inflammatory rheumatic disorder. The crystal structures described here show that a modified self-peptide, pVIPR-U5 (RRKWURWHL; U = citrulline), is presented by the two HLA-B27 molecules in distinct conformations. These binding modes differ not only drastically from each other but also from the conformations exhibited by the non-citrullinated peptide in a given subtype. The differential reactivity of HLA-B27-restricted cytotoxic T cells with modified or unmodified pVIPR supports the structural findings and shows that the presentation of citrullinated peptides has the potential to influence immune responses.     

Clinical characteristics of patients with spondyloarthritides and HLA-B27 positive antigen

The aim of this study was to present our experiences in diagnosing spondyloarthritides (SpA), and to list the most common clinical features of HLA-B27 positive patients. The study included 65 HLA-B27 positive patients with confirmed diagnosis of ankylosing spondylitis (AS) and psoriatic arthritis (PsA) who were analyzed between 2009 and 2010 in Clinic of Internal Medicine in Osijek. The diagnosis of seronegative spondyloarthritides was based on the ASAS (Assessment in AS Working Group) classification criteria for axial and then supplemented with ASAS criteria for peripheral SpA and was confirmed by radiological techniques. For diagnosing the ankylosing spondylitis (AS), there have been applied the modified New York criteria. Radiological criteria for definite sacroiliitis according to the modified New York criteria is bilateral sacroiliitis, grade 2-4 (> or = 2) or unilateral sacroiliitis, grade 3-4. For diagnosing the psoriatic arthritis (PsA), there were used CASPAR diagnostic criteria. Other features of SpA are defined within the existing classification criteria. HLA-B27 antigen was determined by direct immune-fluorescence technique using flow cytometer. The average age of patients was 50.34 years, of whom 27 female (41.53%), 38 male (58.46%). Duration of illness was 15.79 years on average. With 75.38% of patients, there had been determined the diagnosis of AS; 24.62% of patients had the diagnosis of PsA. The most common clinical characteristics that patients had were: inflammatory back pain (pain Inflammation along the lumbosacral spine), peripheral arthritis, intermittent pain in the gluteus, sacroiliitis, enthesitis, uveitis, dactilitis.     

Animal models of HLA-B27-associated diseases

The HLA-B27 molecule is strongly associated with the spondyloarthropathies (SpA), a group of inflammatory conditions affecting the skeleton, the skin and several mucosae. The mechanism of this association remains unknown, largely because the HLA-B27 molecule displays normal function. A disease that closely mimicks SpA arises spontaneously in HLA-B27 transgenic rats. This disease is dependent on the presence of a normal bacterial flora and implicates the immune system. The presence of both CD4+ T cells and antigen presenting cells (APCs) expressing high levels of HLA-B27, seems of critical importance in its pathogenesis, whereas CD8+ T cells are dispensable. The T cell stimulatory function of APCs is disturbed by the HLA-B27 molecule. This disease could result from a failure of tolerance, related in part to high level of B27 expression in professional APCs and to the immune response to gut bacteria. In contrast, HLA-B27 transgenic mice have usually remained healthy. However, two types of inflammatory conditions affecting the skeleton, which arise in mice of susceptible background after exposure to a conventional bacterial flora, are increased by an HLA-B27 transgene. The first is ANKENT, a spontaneous ankylosing enthesitis that affects ankle and/or tarsal joints of ageing mice; the second is a spontaneous arthritis of hindpaws developing in mice lacking endogenous mbeta2m. As in rats, the absence of CD8+ T cells in the latter model, argues against the "arthritogenic peptide" hypothesis. In these mbeta2m0 mice, B27 free heavy chain could be implicated in the pathogenesis of arthritis by presenting extracellular peptides to CD4+ T cells.     

HLA-B27 misfolding and spondyloarthropathies

HLA-B27 plays a central role in the pathogenesis of many spondyloarthropathies and in particular ankylosing spondylitis. The observation that the HLA-B27 heavy chain has a tendency to misfold has raised the possibility that associated diseases may belong in a rapidly expanding category of protein misfolding disorders. The synthesis of the HLA-B27 heavy chain, assembly with β₂m and the loading of peptide cargo, occurs in the endoplasmic reticulum (ER) before transport to the cell surface. The evidence indicates that misfolding occurs in the ER prior to β₂m association and peptide optimization and is manifested in the formation of aberrant inter- and intra-chain disulfide bonds and accumulation of heavy chain bound to the chaperone BiP. Enhanced accumulation of misfolded heavy chains during the induction of class I expression by cytokines, can cause ER stress resulting in activation of the unfolded protein response (UPR).Effects of UPR activation on cytokine production are beginning to emerge and may provide important missing links between HLA-B27 misfolding and spondyloarthritis. In this chapter we will review what has been learned about HLA-B27 misfolding in human cells and in the transgenic rat model of spondyloarthritis-like disease, considering it in the context of other protein misfolding disorders. These studies provide a framework to support much needed translational work assessing HLA-B27 misfolding and UPR activation in patient-derived material, its consequences for disease pathogenesis and ultimately how and where to focus intervention strategies.Key words: ankylosing spondylitis, arthritis, protein misfolding, unfolded protein response, interleukin (IL)-17, cytokines     

The role of HLA-B27 polymorphism and molecular mimicry in spondylarthropathy

Ankylosing spondylitis (AS), reactive arthritis (ReA) and other related spondyloarthropathies (SpAs) are characterized by a strong association with the major histocompatibility complex allele HLA-B27. Experimental evidence from humans and transgenic rodents suggests that HLA-B27 is itself involved in the pathogenesis of SpA. Population and peptide-specificity analysis of HLA-B27 suggest it has a pathogenic function related to antigen presentation. Putative roles for infectious agents have been proposed in ReA and suggested in AS. However, the mechanism by which HLA-B27 and bacteria interact to induce arthritis is not clear. Molecular mimicry between bacterial epitopes that cross-react with self-B27 peptides is the most persuasive explanation for the pathogenesis of SpA. The experimental studies reviewed here have greatly increased our knowledge of the structure, function and disease association of HLA-B27.     

人白细胞抗原HLA-B27在强直性脊柱炎中的临床意义

目的:探讨人白细胞抗原HLA-B27在强直性脊柱炎(AS)中的临床意义,及联合检测HLA-B27、C反应蛋白(CRP)和红细胞沉降率(ESR)的意义。方法:对18例AS患者、24例腰背痛患者和21健康体检者的血液标本进行HLA-B27、CRP、ESR检测,并比较其HLA-B27阳性率。结果:与健康对照组比较,AS组的3项指标均高于对照组(P0.05),且AS组HLA-B27阳性率均高于另外2组(P0.05)。结论:HLA-B27在强直性脊柱炎诊断中具有重要意义,与其具有高度疾病相关性,联合检测HLA-B27及CRP比联合检测HLA-B27及ESR更有利于疾病诊断。     

Rapid Antigen Processing and Presentation of a Protective and Immunodominant HLA-B*27-restricted Hepatitis C Virus-specific CD8+ T-cell Epitope

HLA-B*27 exerts protective effects in hepatitis C virus (HCV) and human immunodeficiency virus (HIV) infections. While the immunological and virological features of HLA-B*27-mediated protection are not fully understood, there is growing evidence that the presentation of specific immunodominant HLA-B*27-restricted CD8+ T-cell epitopes contributes to this phenomenon in both infections. Indeed, protection can be linked to single immunodominant CD8+ T-cell epitopes and functional constraints on escape mutations within these epitopes. To better define the immunological mechanisms underlying HLA-B*27-mediated protection in HCV infection, we analyzed the functional avidity, functional profile, antiviral efficacy and naïve precursor frequency of CD8+ T cells targeting the immunodominant HLA-B*27-restricted HCV-specific epitope as well as its antigen processing and presentation. For comparison, HLA-A*02-restricted HCV-specific epitopes were analyzed. The HLA-B*27-restricted CD8+ T-cell epitope was not superior to epitopes restricted by HLA-A*02 when considering the functional avidity, functional profile, antiviral efficacy or naïve precursor frequency. However, the peptide region containing the HLA-B*27-restricted epitope was degraded extremely fast by both the constitutive proteasome and the immunoproteasome. This efficient proteasomal processing that could be blocked by proteasome inhibitors was highly dependent on the hydrophobic regions flanking the epitope and led to rapid and abundant presentation of the epitope on the cell surface of antigen presenting cells. Our data suggest that rapid antigen processing may be a key immunological feature of this protective and immunodominant HLA-B*27-restricted HCV-specific epitope.     

Identification of novel human aggrecan T cell epitopes in HLA-B27 transgenic mice associated with spondyloarthropathy

The pathology of ankylosing spondylitis, reactive arthritis, and other spondyloarthropathies (SpA) is closely associated with the human leukocyte class I Ag HLA-B27. A characteristic finding in SpA is inflammation of cartilage structures of the joint, in particular at the site of ligament/tendon and bone junction (enthesitis). In this study, we investigated the role of CD8+ T cells in response to the cartilage proteoglycan aggrecan as a potential candidate autoantigen in BALB/c-B27 transgenic mice. We identified four new HLA-B27-restricted nonamer peptides, one of them (no. 67) with a particularly strong T cell immunogenicity. Peptide no. 67 immunization was capable of stimulating HLA-B27-restricted, CD8+ T cells in BALB/c-B27 transgenic animals, but not in wild-type BALB/c mice. The peptide was specifically recognized on P815-B27 transfectants by HLA-B27-restricted CTLs, which were also detectable by HLA tetramer staining ex vivo as well as in situ. Most importantly, analysis of the joints from peptide no. 67-immunized mice induced typical histological signs of SpA. Our data indicate that HLA-B27-restricted epitopes derived from human aggrecan are involved in the induction of inflammation (tenosynovitis), underlining the importance of HLA-B27 in the pathogenesis of SpA.     

Identification of several functional subgroups of HLA-B27 by restriction of the activity of antiviral T killer lymphocytes

Anti-Epstein-Barr virus and antiinfluenza A cytotoxic T lymphocytes (CTL) have been used to study the restriction of human antiviral responses by HLA-B27 antigens. Three functional subgroups of HLA-B27 have been clearly distinguished by this restriction-typing assay. No cross-reaction could be detected between the three subgroups either at the CTL level or at the level of antigen-presenting cells. The cells of subgroup 1 are always positive [M2(+)] when tested in immunofluorescence with a monoclonal B27-specific antibody which divides HLA-B27 into a major M2(+) and a minor M2(–) subgroup. These M2(+) group 1 cells are apparently also HLA-B27W as previously shown by Ivanyi and co-workers using anti-HLA-CTL. Subgroup 2 includes only M2(–) cells. A comparison between this group and the previously described HLA-B27K is not fully conclusive, since two typing cells which were clearly HLA-B27K apparently did not belong to group 2. Only two donors, both of Oriental origin, have been included in subgroup 3. Both of them were M2 intermediate. These results demonstrate (1) the existence of several functional subgroups of HLA-B27 with an interesting correlation with the M2(+), M2(–), or M2 intermediate phenotypes, and (2) the possibility of using the restriction-typing assay to define such functional subgroups not detected by classical allosera.Abbreviations used in this paper CTL cytolytic T lymphocyte - EBV Epstein-Barr virus - AS ankylosing spondylarthritis - PBL peripheral blood leukocytes - MSL medium for separation of lymphocyte - UV ultra violet - PHA-M phytohemagglutinin M - LCL lymphoblastoid cells lines - FCS fetal calf serum - CRT chromium release test - SCR specific chromium release - LU lytic units - MoAb monoclonal antibodies.