Th17 cells expressing KIR3DL2+ and responsive to HLA-B27 homodimers are increased in ankylosing spondylitis

CD4 Th cells producing the proinflammatory cytokine IL-17 (Th17) have been implicated in a number of inflammatory arthritides including the spondyloarthritides. Th17 development is promoted by IL-23. Ankylosing spondylitis, the most common spondyloarthritis (SpA), is genetically associated with both HLA-B27 (B27) and IL-23R polymorphisms; however, the link remains unexplained. We have previously shown that B27 can form H chain dimers (termed B27(2)), which, unlike classical HLA-B27, bind the killer-cell Ig-like receptor KIR3DL2. In this article, we show that B27(2)-expressing APCs stimulate the survival, proliferation, and IL-17 production of KIR3DL2(+) CD4 T cells. KIR3DL2(+) CD4 T cells are expanded and enriched for IL-17 production in the blood and synovial fluid of patients with SpA. Despite KIR3DL2(+) cells comprising a mean of just 15% of CD4 T in the peripheral blood of SpA patients, this subset accounted for 70% of the observed increase in Th17 numbers in SpA patients compared with control subjects. TCR-stimulated peripheral blood KIR3DL2(+) CD4 T cell lines from SpA patients secreted 4-fold more IL-17 than KIR3DL2(+) lines from controls or KIR3DL2(-) CD4 T cells. Strikingly, KIR3DL2(+) CD4 T cells account for the majority of peripheral blood CD4 T cell IL-23R expression and produce more IL-17 in the presence of IL-23. Our findings link HLA-B27 with IL-17 production and suggest new therapeutic strategies in ankylosing spondylitis/SpA.     

Hierarchy in the assembly of HLA-B27 and HLA-Cw3 molecules in transgenic mice

Cell surface expression of human class I molecules in transgenic mice is dependent upon the available pool of ₂-microglobulin (₂m) and the affinity between mouse ₂m and human class I molecules. HLA-B27 and HLA-Cw3 transgenes can be expressed in mouse strains of the H-2 haplotypes b,f,k, and s which encode two endogenous class I genes mapping to H-2K and H-2D. The human class I genes cannot be expressed on H-2 ^dand H-2 ^qhaplotypes which encode three endogenous class I molecules (K,D,L). This suggests that there may be only enough mouse ₂m molecules to support three class I molecules. When both the HLA-B27 and HLA-Cw3 genes are introduced into H-2 ^bmice, only HLA-Cw3 reaches the cell surface. This suggests that HLA-Cw3 has a higher affinity than HLA-B27 for mouse ₂m. The possible implications of our findings regarding the assembly, transport, and expression of class I MHC molecules in vivo are discussed.     

Amyloid-like features of polyglutamine aggregates and their assembly kinetics

The repeat length-dependent tendency of the polyglutamine sequences of certain proteins to form aggregates may underlie the cytotoxicity of these sequences in expanded CAG repeat diseases such as Huntington's disease. We report here a number of features of various polyglutamine (polyGln) aggregates and their assembly pathways that bear a resemblance to generally recognized defining features of amyloid fibrils. PolyGln aggregation kinetics displays concentration and length dependence and a lag phase that can be abbreviated by seeding. PolyGln aggregates exhibit classical beta-sheet-rich circular dichroism spectra consistent with an amyloid-like substructure. The fundamental structural unit of all the in vitro aggregates described here is a filament about 3 nm in width, resembling the protofibrillar intermediates in amyloid fibril assembly. We observed these filamentous structures either as isolated threads, as components of ribbonlike sheets, or, rarely, in amyloid-like twisted fibrils. All of the polyGln aggregates described here bind thioflavin T and shift its fluorescence spectrum. Although all polyGln aggregates tested bind the dye Congo red, only aggregates of a relatively long polyGln peptide exhibit Congo red birefringence, and this birefringence is only observed in a small portion of these aggregates. Remarkably, a monoclonal antibody with high selectivity for a generic amyloid fibril conformational epitope is capable of binding polyGln aggregates. Thus, polyGln aggregates exhibit most of the characteristic features of amyloid, but the twisted fibril structure with Congo red birefringence is not the predominant form in the polyGln repeat length range studied here. We also find that polyGln peptides exhibit an unusual freezing-dependent aggregation that appears to be caused by the freeze concentration of peptide and/or buffer components. This is of both fundamental and practical significance. PolyGln aggregation is revealed to be a highly specific process consistent with a significant degree of order in the molecular structure of the product. This ordered structure, or the assembly process leading to it, may be responsible for the cell-specific neuronal degeneration observed in Huntington's and other expanded CAG repeat diseases.     

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     

HLA-B27 homodimers and free H chains are stronger ligands for leukocyte Ig-like receptor B2 than classical HLA class I

Possession of HLA-B27 (B27) strongly predisposes to the development of spondyloarthritis. B27 forms classical heterotrimeric complexes with β(2)-microglobulin (β2m) and peptide and (β2m free) free H chain (FHC) forms including B27 dimers (termed B27(2)) at the cell surface. In this study, we characterize the interaction of HLA-B27 with LILR, leukocyte Ig-like receptor (LILR)B1 and LILRB2 immune receptors biophysically, biochemically, and by FACS staining. LILRB1 bound to B27 heterotrimers with a K(D) of 5.3 ± 1.5 μM but did not bind B27 FHC. LILRB2 bound to B27(2) and B27 FHC and B27 heterotrimers with K(D)s of 2.5, 2.6, and 22 ± 6 μM, respectively. Domain exchange experiments showed that B27(2) bound to the two membrane distal Ig-like domains of LILRB2. In FACS staining experiments, B27 dimer protein and tetramers stained LILRB2 transfectants five times more strongly than B27 heterotrimers. Moreover, LILRB2Fc bound to dimeric and other B27 FHC forms on B27-expressing cell lines more strongly than other HLA-class 1 FHCs. B27-transfected cells expressing B27 dimers and FHC inhibited IL-2 production by LILRB2-expressing reporter cells to a greater extent than control HLA class I transfectants. B27 heterotrimers complexed with the L6M variant of the GAG KK10 epitope bound with a similar affinity to complexes with the wild-type KK10 epitope (with K(D)s of 15.0 ± 0.8 and 16.0 ± 2.0 μM, respectively). Disulfide-dependent B27 H chain dimers and multimers are stronger ligands for LILRB2 than HLA class I heterotrimers and H chains. The stronger interaction of B27 dimers and FHC forms with LILRB2 compared with other HLA class I could play a role in spondyloarthritis pathogenesis.     

Immunochemical variants of HLA-B27

Detailed study of HLA-B27 was prompted by the extremely strong associations between this antigen and spondyloarthropathies. Despite the relative homogeneity of this antigen when defined by alloantisera, B27 reactivity with the monoclonal antibody B27M2 suggests previously unrecognized heterogeneity. To define and confirm this heterogeneity on a molecular level, detergent extracts were prepared from B cell lines derived from individuals reactive (+) or unreactive (-) with the B27M2 antibody. Extracts were immunoprecipitated by specific allogeneic or monoclonal antibodies and analyzed by two-dimensional polyacrylamide gel electrophoresis. By this method the B27M2+ and B27M2- variants of HLA-B27 had different isoelectric points (pl) and could be distinguished from each other and from a different (Bw44) control alloantigen. Blockade of glycosylation by pretreatment of cells with tunicamycin did not alter pl but did reduce HLA antigens by approximately 3000 daltons. These data demonstrate that B27 antigens can be subdivided into subsets with different molecular composition. The effects of this heterogeneity upon the associations of B27 and disease are not yet known.     

Identification of HLA-B27-restricted peptides from the Chlamydia trachomatis proteome with possible relevance to HLA-B27-associated diseases

The association of HLA-B27 with ankylosing spondylitis and reactive arthritis is the strongest one known between an MHC class I Ag and a disease. We have searched the proteome of the bacterium Chlamydia trachomatis for HLA-B27 binding peptides that are stimulatory for CD8(+) cells both in a model of HLA-B27 transgenic mice and in patients. This was done by combining two biomathematical computer programs, the first of which predicts HLA-B27 peptide binding epitopes, and the second the probability of HLA-B27 peptide generation by the proteasome system. After preselection, immunodominant peptides were identified by Ag-specific flow cytometry. Using this approach we have identified for the first time nine peptides derived from different C. trachomatis proteins that are stimulatory for CD8(+) T cells. Eight of these nine murine-derived peptides were recognized by cytotoxic T cells. The same strategy was used to identify B27-restricted chlamydial peptides in three patients with reactive arthritis. Eleven peptides were found to be stimulatory for patient-derived CD8(+) T cells, of which eight overlapped those found in mice. Additionally, we applied the tetramer technology, showing that a B27/chlamydial peptide containing one of the chlamydial peptides stained CD8(+) T cells in patients with Chlamydia-induced arthritis. This comprehensive approach offers the possibility of clarifying the pathogenesis of B27-associated diseases.     

HLA-B27-Homodimer-Specific Antibody Modulates the Expansion of Pro-Inflammatory T-Cells in HLA-B27 Transgenic Rats

Objectives

HLA-B27 is a common genetic risk factor for the development of Spondyloarthritides (SpA). HLA-B27 can misfold to form cell-surface heavy chain homodimers (B27₂) and induce pro-inflammatory responses that may lead to SpA pathogenesis. The presence of B27₂ can be detected on leukocytes of HLA-B27+ Ankylosing spondylitis (AS) patients and HLA-B27 transgenic rats. We characterized a novel B27₂–specific monoclonal antibody to study its therapeutic use in HLA-B27 associated disorders.

Methods

The monoclonal HD5 antibody was selected from a phage library to target cell-surface B27₂ homodimers and characterized for affinity, specificity and ligand binding. The immune modulating effect of HD5 was tested in HLA-B27 transgenic rats. Onset and progression of disease profiles were monitored during therapy. Cell-surface B27₂ and expansion of pro-inflammatory cells from blood, spleen and draining lymph nodes were assessed by flow cytometry.

Results

HD5 bound B27₂ with high specificity and affinity (K^d = 0.32 nM). HD5 blocked cell-surface interaction of B27₂ with immune regulatory receptors KIR3DL2, LILRB2 and Pirb. In addition, HD5 modulated the production of TNF from CD4+ T-cells by limiting B27₂ interactions in vitro. In an HLA-B27 transgenic rat model repetitive dosing of HD5 reduced the expansion of pro-inflammatory CD4+ T-cells, and decreased the levels of soluble TNF and number of cell-surface B27₂ molecules.

Conclusion

HD5 predominantly inhibits early TNF production and expansion of pro-inflammatory CD4+ T-cells in HLA-B27 transgenic rats. Monoclonal antibodies targeting cell-surface B27₂ propose a new concept for the modulation of inflammatory responses in HLA-B27 related disorders.     

Molecular typing of HLA-B27 alleles

HLA-B27 represents a family of closely related antigens. Six alleles which differ in a limited number of nucleotide substitutions have been described (B*2701—B*2706). These changes are clustered in 1 and 2 domains. Polymerase chain reaction strategies were designed to amplify specific regions of class I exons 2 and 3. Amplified sequences were tested with eight sequence-specific oligonucleotides to distinguish all B27 subtypes. We also subtyped B27 in 50 healthy Spanish individuals using this procedure. The B*2705 subtype is over-represented in our population (96%). The remaining 4% carried the B*2702 allele. This finding is in agreement with the frequencies described by other techniques (cytotoxic T lymphocytes and isoeletric-focusing) for Caucasian populations. Class I oligotyping is a poorly developed field with significant potential applications. This procedure of genotyping B27 alleles is a reliable method which can be used in transplantation and B27-associated disease studies.     

Non-antigen presenting effects of HLA-B27

Spondyloarthropathies (SpA) are a group of chronic rheumatic diseases, which show a strong asoociation with human leukocyte antigen (HLA)-B27. Although the association between HLA-B27 and the susceptibility to SpA was discovered thirty years ago, the exact mechanism by which HLA-B27 predisposes to disease development remains unclear. The classical role of MHC class I molecules is to present peptides for CD8+ T cells. Therefore, it has been proposed that the antigen presenting function of HLA-B27 is somehow altered in the patients developing SpA. However, despite extensive research, the attempts to create a comprehensive theory that would explain the role of HLA-B27 as an antigen presenting molecule in the development of SpA have been unsuccessful. Reactive arthritis (ReA) belongs to the group of SpA. It is a joint inflammation developing after certain bacterial infections e.g. Salmonella, Yersinia, and Chlamydia. Several unrelated observations indicate that HLA-B27 modulates the interaction between ReA-triggering bacteria and host cell. These findings suggest that HLA-B27 may possess functions, which are unrelated to antigen presentation. In this paper, we summarize these findings and discuss their potential impact in the development of SpA.     

Epitope mapping of HLA-B27 and HLA-B7 antigens by using intradomain recombinants

To study the HLA-B7 and HLA-B27 antigenic determinants, hybrid genes between these two alleles were constructed by in vivo recombination in Escherichia coli. After transfection of these genes into P815 (high transfection efficiency recipient) murine cells, the bindings of Bw6, HLA-B7, and HLA-B27 allele-specific mAb were studied, as well as that of human anti-HLA-B7 and anti-HLA-B27 monospecific alloantisera. Most of the HLA-B7 antigenic determinants were assigned to the first external domain of the molecule. Four different epitopic areas could be defined: the Bw6 epitope was associated with residues 82 and 83; the BB7.1 epitope to amino acids 63, 67, and 70; the MB40.2 and MB40.3 epitope to amino acid sequence 177-180, and human alloantisera identified as an epitope associated with residue 9. HLA-B27 antigenicity studied by TM-1 mAb was found to involve residues 77 and 80 in the alpha-1 domain. Results obtained with human monospecific alloantisera allowed the definition of an additional allospecific site associated with the NH2 terminal part on the alpha-1 domain of HLA-B27. Epitope mapping fits with data obtained by sequence comparisons and is discussed with reference to the crystallographic three-dimensional structure of the HLA-A2 molecule.     

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.     

Molecular analysis of a functional subtype of HLA-B27. A possible evolutionary pathway for HLA-B27 polymorphism

The structure of a new HLA-B27 subtype antigen B27.4(B27D), distinguishable from the HLA-B27.1, B27.2, and B27.3 subtypes by cytolytic T lymphocytes and isoelectric focusing, has been established by comparative peptide mapping and sequence analysis. HLA-B27.4 differs from the main B27.1 subtype in the same two changes of aspartate-77 to serine-77 and valine-152 to glutamate-152, which distinguish the B27.1 and B27.3 subtypes. In addition, there are two other amino acid changes of histidine-114 to aspartate-114 and of aspartate-116 to tyrosine-116, which are unique to B27.4. The close structural relationship between B27.3 and B27.4 explains the similarity of these two subtypes in terms of T cell recognition. The presence of the two single amino acid differences between B27.3 and B27.4 within a span of three residues in the linear sequence provides a new example, suggesting that gene conversion-like mechanisms play a major role in the diversification of HLA-B27. A comparison of the structure of HLA-B27.4 with those of B27.1, B27.2, and B27.3 in the context of their ethnic distribution suggests that the diversification of the HLA-B27 antigens is an ongoing process that has continued after the separation of the major ethnic groups. A tentative evolutionary model for HLA-B27 polymorphism is proposed.     

A high-resolution polymerase chain reaction-sequence-specific primer HLA-B*27 typing set and its application in routine HLA-B27 testing

We designed a set of 35 polymerase chain reaction sequence-specific primers (PCR-SSP) in 29 SSP mixtures to assign 29 HLA-B*27 4-digit level alleles (B*2701-B*2721 and B*2723-B*2730). This was used in conjunction with our 41 PCR-SSP primer mixture low-resolution HLA-B typing set to fully differentiate B*27 from all other HLA-B alleles. Successful typing set validation used 521 B*27 samples covering 13 (B*2701-B*2710 and B*2712, B*2717, B*2723) alleles. The distribution of B*27 alleles was determined in a random population of 4020 local blood donors and the use of PCR-SSP B*27 typing in our routine flow cytometry-based HLA-B27/B2708 typing strategy is described.     

HLA-B27 antigenicity: antibodies against the chemically synthesized 63-84 peptide from HLA-B27.1 display alloantigenic specificity and discriminate among HLA-B27 subtypes

A chemically synthesized peptide with an amino acid sequence identical to that of the segment spanning residue 63-84 of the major HLA-B27.1 subtype antigen has been obtained. Specific antibodies were raised in rabbits against this peptide, coupled to keyhole limpet hemocyanin carrier. These antibodies lysed lymphoblastoid cell lines expressing HLA-B27.1 in a complement-mediated cytotoxicity assay. They lysed neither B27-negative target cells, nor B27-positive cells expressing other B27 subtype antigens. Complement-mediated lysis of B27.1-positive targets was inhibited by free peptide and by peptide coupled to an unrelated carrier. In addition, the lytic action of the rabbit antiserum was blocked by a monoclonal antibody with no complement-activating capacity that under the conditions of the assay, was specific for HLA-B27. These results indicate that rabbit antibodies against the 63-84 peptide recognize the native HLA-B27.1 antigen; this antiserum is allospecific in character; and it discriminates among B27 subtypes. Thus the data provide direct evidence on the contribution of the hypervariable region spanning residues 63-84 to the alloantigenic specificity of HLA-B27.     

Structural Basis for T Cell Alloreactivity among Three HLA-B14 and HLA-B27 Antigens

The existence of cytotoxic T cells (CTL) cross-reacting with the human major histocompatibility antigens HLA-B14 and HLA-B27 suggests that their alloreactivity could be due to presentation of shared peptides in similar binding modes by these molecules. We therefore determined the crystal structures of the subtypes HLA-B*1402, HLA-B*2705, and HLA-B*2709 in complex with a proven self-ligand, pCatA (peptide with the sequence IRAAPPPLF derived from cathepsin A (residues 2–10)), and of HLA-B*1402 in complex with a viral peptide, pLMP2 (RRRWRRLTV, derived from latent membrane protein 2 (residues 236–244) of Epstein-Barr virus). Despite the exchange of 18 residues within the binding grooves of HLA-B*1402 and HLA-B*2705 or HLA-B*2709, the pCatA peptide is presented in nearly identical conformations. However, pLMP2 is displayed by HLA-B*1402 in a conformation distinct from those previously found in the two HLA-B27 subtypes. In addition, the complexes of HLA-B*1402 with the two peptides reveal a nonstandard, tetragonal mode of the peptide N terminus anchoring in the binding groove because of the exchange of the common Tyr-171 by His-171 of the HLA-B*1402 heavy chain. This exchange appears also responsible for reduced stability of HLA-B14-peptide complexes in vivo and slow assembly in vitro. The studies with the pCatA peptide uncover that CTL cross-reactive between HLA-B14 and HLA-B27 might primarily recognize the common structural features of the bound peptide, thus neglecting amino acid replacements within the rim of the binding grooves. In contrast, structural alterations between the three complexes with the pLMP2 peptide indicate how heavy chain polymorphisms can influence peptide display and prevent CTL cross-reactivity between HLA-B14 and HLA-B27 antigens.T cells possessing the ability to recognize major histocompatibility complex (MHC)² molecules from another individual of the same species, also termed alloreactive T cells, may constitute up to 10% of the T cell pool of an individual, and their precursor frequency can be 100–1,000-fold higher than that of self-restricted T cells directed against a foreign peptide (1, 2). The ability of alloreactive T cells to cross-react with nonself-MHC molecules is a major obstacle preventing successful organ transplantations (3–5). Two mechanisms, direct or indirect allorecognition, can be responsible for the rejection of a transplant by alloreactive T cells (6). In the first case, donor cells expressing MHC molecules are directly recognized by host T cells (7), whereas indirect allorecognition involves the presentation of peptides derived from donor proteins by MHC molecules of the host, followed by the detection of the complexes by the host T cells (8). However, although alloreactive T cells are very common and of great clinical importance, neither the primary basis for their existence nor the reasons underlying their cross-reactivity are sufficiently understood to draw general conclusions (9–11). Only very few studies have addressed the structural basis for the recognition of distinct MHC antigens by cross-reactive T cells (12–18). One of the most important questions regards the individual contribution of the bound peptide and binding groove residues of the heavy chain (HC) of MHC class I antigens to the interaction with T cell receptors (TCR).Here we analyze an HLA-B14 subtype, HLA-B*1402 (named B*1402), as well as two HLA-B27 subtypes, HLA-B*2705 and HLA-B*2709 (named B*2705 and B*2709), to shed light on the structural basis of peptide presentation and T cell alloreactivity among these HLA-B molecules. The amino acid sequences of B*1402 and B*2705 HC differ from each other at 18 positions, all of which are part of the peptide-binding groove (Fig. 1). These amino acid exchanges result in different repertoires of bound peptides; B*1402 and B*2705 share only about 4% of their peptides (19), whereas this value rises to 88% for the B*2705 and B*2709 subtypes (20), which are distinguished only by a single residue at the floor of the binding groove (B*2705, Asp-116; B*2709, His-116). The structural similarities between the two HLA-B27 subtypes (21–27) permit extensive cross-reactivity (up to 90%) of cytotoxic T cells (CTL) (28), whereas CTL alloreactivity between B*1402 and B*2705 is drastically reduced (to about 3%) (19), in line with the very limited overlap of their peptide repertoires.Open in a separate windowFIGURE 1.Amino acid sequence differences among B*1402 and B*2705 HC. The 18 residues distinguishing the two subtypes are all located in or in the immediate vicinity of the peptide-binding groove. B*2705 differs from B*2709 only by a D116H exchange (not shown). The residues are indicated by spheres with volumes roughly proportional to the volumes of the respective amino acid side chain in solution (77). The spheres are colored according to the biochemical properties of the respective amino acids, as indicated at the bottom of the image.The HLA-B14 and HLA-B27 subtypes are distinguished from most other HLA class I molecules in their requirement for an arginine at anchor position 2 of the bound peptide (p2) (20, 29, 30). This preference is nearly absolute in B*2705 and B*2709 (31), whereas B*1402 tolerates also glutamine, glutamate, and proline as p2 anchors (19, 29). Statistically significant differences between B*1402 and B*2705 are also found at several other peptide positions (19). Previous structural and cellular studies of the HLA-B27 subtypes have suggested that molecular mimicry between the viral peptide pLMP2 (RRRWRRLTV, derived from Epstein-Barr virus latent membrane protein 2, residues 236–244) and the self-peptide pVIPR (RRKWRRWHL, derived from vasoactive intestinal peptide type 1 receptor, residues 400–408), when bound to B*2705, serves as an example of how a cellular immune response could be triggered that might contribute to the onset of ankylosing spondylitis (AS) through an autoimmune mechanism (22, 24). CTL that recognize the B*2705 and the B*2709 subtypes in complex with the self-peptide pVIPR (22) exemplify alloreactivity in this system, although the D116H micropolymorphism is deeply buried and not directly accessible to a TCR.Alloreactive T cells are known to recognize a very diverse array of alloantigen-bound peptides (32, 33), so that virtually each T cell clone can be assumed to be specific for a distinct peptide. For this reason, the substantial correlation found in previous studies between peptide and the alloreactive T cell epitope sharing among HLA-B27 (reviewed in Ref. 34) or HLA-B14 subtypes (only 28.4% partial or full cross-reactivity, similar to peptide overlapping between the subtypes B*1402 and B*1403, see Ref. 19) supports a prominent role of peptides in determining alloreactive T cell cross-reaction, and it suggests that many shared ligands adopt antigenically similar conformations when bound to distinct HLA-B molecules. On the other hand, the results reported by Merino et al. (19) also demonstrate that the few CTL that cross-react with B*1402 and B*2705 did not exhibit cross-reactivity with B*1403, which is distinguished from B*1402 only by a single amino acid exchange in the α2-helix. Furthermore, they show that alloreactive CTL from various donors directed against B*2705 did not lyse cells expressing either B*1402 or B*1403, although the number of CTL tested might not have been high enough to detect a presumably low degree of cross-reactivity. Without structural data from HLA-B14 subtypes, however, these results are difficult to interpret.The pCatA peptide (IRAAPPPLF, derived from the signal sequence of cathepsin A, residues 2–10) is among the very few known common ligands of B*1402, B*2705 (19), and B*2709³ and can thus serve to study how a very different (B*1402) and two very similar subtypes (B*2705 and B*2709) handle a common ligand. On the other hand, the pLMP2 peptide is a proven natural ligand only of B*2705, whose possible presentation in vivo by B*2709 and HLA-B14 is not yet known, although this peptide can be complexed in vitro with B*2709 (24) and also with B*1402 (35). From previous crystallographic studies, it was known that pLMP2 is presented by the two HLA-B27 antigens in very different conformations (24). We expected that the pronounced sequence differences between B*1402 and the HLA-B27 alloantigens (Fig. 1) might even enhance the conformational dissimilarities that are observed when two very closely related subtypes such as B*2705 and B*2709 are compared. Discrepancies in peptide display could reasonably be expected to prevent CTL cross-reaction, so that pLMP2 might be considered as a representative of the vast majority of HLA-B14- and HLA-B27-presented ligands that must be responsible for the low degree of CTL cross-reactivity between these alloantigens. Despite these presumed differences between pCatA and pLMP2, both peptides may be seen as examples of ligands that could principally allow direct allorecognition.Here we report the crystal structures of B*1402·pCatA, B*2705·pCatA, B*2709·pCatA, and B*1402·pLMP2, and we compare them with each other and with the previously reported structures of B*2705·pLMP2 and B*2709·pLMP2 (24).     

Structure and diversity of HLA-B27-specific T cell epitopes. Analysis with site-directed mutants mimicking HLA-B27 subtype polymorphism.

HLA-B27 subtype polymorphism is amenable to differential recognition by CTL. Site-directed mutagenesis was used to construct a series of HLA-B27 mutants reproducing most of the changes occurring in the natural subtypes. The reactivity of 21 anti-HLA-B27 CTL clones was examined with these mutants to address three issues concerning the alloreactive response against HLA-B27: 1) diversity of clonotypic specificities, 2) structural features of the epitopes recognized by these clones, and 3) role of individual positions in the differential recognition of HLA-B27 subtypes. Virtually all CTL clones displayed unique reaction patterns with the mutants, indicating a corresponding diversity of epitopes. However, these share some molecular features, such as certain amino acid residues and related locations. Individual mutations induced complex effects on multiple B27-specific CTL epitopes, revealing some of their very precise stereochemical constrains. An important feature of HLA-B27 subtype polymorphism is that every individual change was relevant, altering recognition by many CTL clones. Although the specific set affected by each mutation was partially different, the global number of clones affected by most changes was very similar. This suggests that the antigenic profile of any given subtype is not dominated by one particular change but is uniquely defined by its corresponding set of changes. An exception was the change at position 152, which totally abrogated recognition by all 20 anti-B*2705 CTL clones. This effect decisively influences the profound differences in T cell recognition between B*2705 and the two subtypes, B*2704 and B*2706, carrying this change. The results are compatible with the idea that HLA-B27 allorecognition may involve multiple peptides bound to the alloantigen on the cell surface.