Novel HLA-B27-restricted Epitopes from Chlamydia trachomatis Generated upon Endogenous Processing of Bacterial Proteins Suggest a Role of Molecular Mimicry in Reactive Arthritis

Reactive arthritis (ReA) is an HLA-B27-associated spondyloarthropathy that is triggered by diverse bacteria, including Chlamydia trachomatis, a frequent intracellular parasite. HLA-B27-restricted T-cell responses are elicited against this bacterium in ReA patients, but their pathogenetic significance, autoimmune potential, and relevant epitopes are unknown. High resolution and sensitivity mass spectrometry was used to identify HLA-B27 ligands endogenously processed and presented by HLA-B27 from three chlamydial proteins for which T-cell epitopes were predicted. Fusion protein constructs of ClpC, Na⁺-translocating NADH-quinone reductase subunit A, and DNA primase were expressed in HLA-B27⁺ cells, and their HLA-B27-bound peptidomes were searched for endogenous bacterial ligands. A non-predicted peptide, distinct from the predicted T-cell epitope, was identified from ClpC. A peptide recognized by T-cells in vitro, NQRA(330–338), was detected from the reductase subunit. This is the second HLA-B27-restricted T-cell epitope from C. trachomatis with relevance in ReA demonstrated to be processed and presented in live cells. A novel peptide from the DNA primase, DNAP(211–223), was also found. This was a larger variant of a known epitope and was highly homologous to a self-derived natural ligand of HLA-B27. All three bacterial peptides showed high homology with human sequences containing the binding motif of HLA-B27. Molecular dynamics simulations further showed a striking conformational similarity between DNAP(211–223) and its homologous and much more flexible human-derived HLA-B27 ligand. The results suggest that molecular mimicry between HLA-B27-restricted bacterial and self-derived epitopes is frequent and may play a role in ReA.     

Molecular mimicry of an HLA-B27-derived ligand of arthritis-linked subtypes with chlamydial proteins

HLA-B27 is strongly associated with spondyloarthropathies, including ankylosing spondylitis and reactive arthritis. The latter disease is triggered by various Gram-negative bacteria. A dodecamer derived from the intracytoplasmic tail of HLA-B27 was a natural ligand of three disease-associated subtypes (B*2702, B*2704, and B*2705) but not of two (B*2706 and B*2709), weakly or not associated to spondyloarthropathy. This peptide was strikingly homologous to protein sequences from arthritogenic bacteria, particularly to a region of the DNA primase from Chlamydia trachomatis. A synthetic peptide with this bacterial sequence bound in vitro disease-associated subtypes equally as the natural B27-derived ligand. The chlamydial peptide was generated by the 20 S proteasome from a synthetic 28-mer with the sequence of the corresponding region of the bacterial DNA primase. Molecular modeling suggested that the B27-derived and chlamydial peptides adopt very similar conformations in complex with B*2705. The results demonstrate that an HLA-B27-derived peptide mimicking arthritogenic bacterial sequences is a natural ligand of disease-associated HLA-B27 subtypes and suggest that the homologous chlamydial peptide might be presented by HLA-B27 on Chlamydia-infected cells.     

Species-specific differences in proteasomal processing and tapasin-mediated loading influence peptide presentation by HLA-B27 in murine cells

Expression of HLA-B27 in murine cells has been used to establish animal models for human spondyloarthritis and for antigen presentation studies, but the effects of xenogeneic HLA-B27 expression on peptide presentation are little known. The issue was addressed in this study. HLA-B27-bound peptide repertoires from human and murine cells overlapped by 75-85%, indicating that many endogenous HLA-B27 ligands are generated and presented in both species. Of 20 differentially presented peptides that were sequenced, only 40% arose from obvious inter-species protein polymorphism, suggesting that differences in antigen processing-loading accounted for many species-specific ligands. Digestion of synthetic substrates with human and murine 20 S proteasomes revealed cleavage differences that accounted for or correlated with differential expression of particular peptides. One HLA-B27 ligand found only in human cells was similarly generated in vitro by human and murine proteasomes. Differential presentation correlated with significantly decreased amounts of this ligand in human tapasin-deficient cells reconstituted with murine tapasin, indicating that species-specific interactions between HLA-B27, tapasin, and/or other proteins in the peptide-loading complex influenced presentation of this peptide. Our results indicate that differences in proteasomal specificity and in interactions involving tapasin determine differential processing and presentation of a significant number of HLA-B27 ligands in human and murine cells.     

Endogenous Processing and Presentation of T-cell Epitopes from Chlamydia trachomatis with Relevance in HLA-B27-associated Reactive Arthritis

Chlamydia trachomatis triggers reactive arthritis, a spondyloarthropathy linked to the human major histocompatibility complex molecule HLA-B27, through an unknown mechanism that might involve molecular mimicry between chlamydial and self-derived HLA-B27 ligands. Chlamydia-specific CD8⁺ T-cells are found in reactive arthritis patients, but the immunogenic epitopes are unknown. A previous screening of the chlamydial genome for putative HLA-B27 ligands predicted multiple peptides that were recognized in vitro by CD8⁺ T-lymphocytes from patients. Here stable transfectants expressing bacterial fusion proteins in human cells were generated to investigate the endogenous processing and presentation by HLA-B27 of two such epitopes through comparative immunoproteomics of HLA-B27-bound peptide repertoires. A predicted T-cell epitope, from the CT610 gene product, was presented by HLA-B27. This is, to our knowledge, the first endogenously processed epitope involved in HLA-B27-restricted responses against C. trachomatis in reactive arthritis. A second predicted epitope, from the CT634 gene product, was not detected. Instead a non-predicted nonamer from the same protein was identified. Both bacterial peptides showed very high homology with human sequences containing the HLA-B27 binding motif. Thus, expression and intracellular processing of chlamydial proteins into human cells allowed us to identify two bacterial HLA-B27 ligands, including the first endogenous T-cell epitope from C. trachomatis involved in spondyloarthropathy. That human proteins contain sequences mimicking chlamydial T-cell epitopes suggests a basis for an autoimmune component of Chlamydia-induced HLA-B27-associated disease.Chlamydia trachomatis is an obligate intracellular parasite that infects the urogenital epithelium. It is a very common pathogen and one frequently inducing reactive arthritis (ReA)¹ (1). Multiple strategies, including down-regulation of major histocompatibility complex (MHC) class I and class II expression (2–4) and persistence, have been developed by the bacteria to evade the immune system. Yet both CD4⁺ and CD8⁺ T-cell responses are activated upon infection (5). In particular, HLA-B27-restricted CD8⁺ T-lymphocytes are found in patients with Chlamydia-induced ReA (6, 7). The role of these cells in the pathogenesis and evolution of ReA to chronic disease is probably mediated by IFN-γ. Secretion of this cytokine is critical for the protective immunity against Chlamydia (8) because it inhibits the bacterial growth (9). However, this is often insufficient to promote complete clearance of C. trachomatis, and actually IFN-γ-induced depletion of the tryptophan pool induces the differentiation of the metabolically active reticular bodies to persistent forms (10), which sustain chronic infection and ReA. The high prevalence of HLA-B27 among patients with Chlamydia-induced ReA (11), especially in its chronic form, suggests a pathogenetic mechanism based on interactive effects of the bacteria and HLA-B27 that seems unrelated to the capacity of C. trachomatis to infect or replicate into HLA-B27-positive cells (12). One such mechanism could be T-cell-mediated autoimmunity elicited by molecular/antigenic mimicry between chlamydial and self-derived HLA-B27 ligands. Antigenic mimicry between chlamydial and homologous α-myosin-derived peptides is crucial to inducing autoimmune myocarditis in mice (13). Breakdown of cytotoxic T-lymphocyte (CTL) tolerance to HLA-B27 was observed in transgenic rats upon exposure to C. trachomatis (14). Cross-reactivity between HLA-B27-restricted Chlamydia-specific CTL with self-derived HLA-B27 epitopes has not been reported. However, a biochemical basis for it was provided by the finding of an endogenously processed and presented peptide from the DNA primase of C. trachomatis with high homology to a self-derived HLA-B27 ligand (15, 16).Because of the likely involvement of HLA-B27 in the pathogenesis of chronically evolving ReA, the role of CD8⁺ T-cell responses in the protective immunity against C. trachomatis and the presence of HLA-B27-restricted T-cells in patients with Chlamydia-induced ReA, the identification of relevant chlamydial epitopes becomes crucial to establish the pathogenetic mechanism of this disease. Unfortunately a direct analysis of chlamydial HLA-B27 ligands expressed on infected cells is exceedingly difficult because of their extremely low amounts, which challenge even the most sensitive techniques of MS. In the case of Chlamydia, the situation is further complicated by the down-regulation of MHC class I expression shortly after infection (3, 4). To our knowledge, only one MHC class I ligand was recently identified, in the mouse system, from Chlamydia muridarum-infected cells using state-of-the-art MS techniques (17). Due in part to this difficulty, alternative approaches, such as expression cloning and synthetic peptide epitope mapping (18, 19) or MHC class I tetramer arrays (20), have been used to identify MHC class I-restricted chlamydial T-cell epitopes in mice. In a previous study (6) predictive algorithms were used to screen the whole genome of C. trachomatis for nonamer peptide sequences containing the HLA-B*2705 binding motif and a high probability of being generated by proteasomal cleavage. This led to identifying multiple sequences that, when used as synthetic peptides in vitro, stimulated CD8⁺ T-cells from patients with Chlamydia-induced ReA. Such cells could also be detected in the synovial fluid of these patients using HLA-B27 tetramers complexed to some of these peptides (7).Although these strategies identify chlamydial sequences that are recognized by CD8⁺ T-cells they do not prove that these peptides are the endogenously processed epitopes that activated the natural T-cell responses to the bacteria in vivo. Because of the intrinsic cross-reactivity of T-cells (21, 22), it is conceivable that synthetic peptides recognized in vitro may be different from the natural epitopes generated by endogenous processing of the chlamydial proteins that elicit the HLA-B27-restricted T-cell responses in ReA patients. To investigate this issue we focused on two predicted epitopes (6). Stable transfectants expressing the corresponding chlamydial proteins fused to green fluorescent protein (GFP) were generated in a B*2705-positive cell line. The endogenous processing and presentation of the predicted epitopes or other peptides from the same bacterial protein were analyzed by comparative immunoproteomics analysis of the B*2705-bound peptide repertoires from transfected and untransfected cells and sequencing of peptides differentially presented on the bacterial protein transfectant.     

A Common Minimal Motif for the Ligands of HLA-B*27 Class I Molecules

CD8⁺ T cells identify and kill infected cells through the specific recognition of short viral antigens bound to human major histocompatibility complex (HLA) class I molecules. The colossal number of polymorphisms in HLA molecules makes it essential to characterize the antigen-presenting properties common to large HLA families or supertypes. In this context, the HLA-B*27 family comprising at least 100 different alleles, some of them widely distributed in the human population, is involved in the cellular immune response against pathogens and also associated to autoimmune spondyloarthritis being thus a relevant target of study. To this end, HLA binding assays performed using nine HLA-B*2705-restricted ligands endogenously processed and presented in virus-infected cells revealed a common minimal peptide motif for efficient binding to the HLA-B*27 family. The motif was independently confirmed using four unrelated peptides. This experimental approach, which could be easily transferred to other HLA class I families and supertypes, has implications for the validation of new bioinformatics tools in the functional clustering of HLA molecules, for the identification of antiviral cytotoxic T lymphocyte responses, and for future vaccine development.     

Identification of novel HLA-B27 ligands derived from polymorphic regions of its own or other class I molecules based on direct generation by 20 S proteasome.

HLA-B27 is strongly associated with ankylosing spondylitis. Natural HLA-B27 ligands derived from polymorphic regions of its own or other class I HLA molecules might be involved in autoimmunity or provide diversity among HLA-B27-bound peptide repertoires from individuals. In particular, an 11-mer spanning HLA-B27 residues 169-179 is a natural HLA-B27 ligand with homology to proteins from Gram-negative bacteria. Proteasomal digestion of synthetic substrates demonstrated direct generation of the B27-(169-179) ligand. Cleavage after residue 181 generated a B27-(169-181) 13-mer that was subsequently found as a natural ligand of B*2705 and B*2704. Its binding to HLA-B27 subtypes in vivo correlated better than B27-(169-179) with association to spondyloarthropathy. Proteasomal cleavage generated also a peptide spanning B*2705 residues 150-158. This region is polymorphic among HLA-B27 subtypes and class I HLA antigens. The peptide was a natural B*2704 ligand. Since this subtype differs from B*2705 at residue 152, it was concluded that the ligand arose from HLA-B*3503, synthesized in the cells used as a source for B*2704-bound peptides. Thus, polymorphic HLA-B27 ligands derived from HLA-B27 or other class I molecules are directly produced by the 20 S proteasome in vitro, and this can be used for identification of such ligands in the constitutive HLA-B27-bound peptide pool.     

Concerted In Vitro Trimming of Viral HLA-B27-Restricted Ligands by Human ERAP1 and ERAP2 Aminopeptidases

In the classical human leukocyte antigen (HLA) class I antigen processing and presentation pathway, the antigenic peptides are generated from viral proteins by multiple proteolytic cleavages of the proteasome (and in some cases other cytosolic proteases) and transported to the endoplasmic reticulum (ER) lumen where they are exposed to aminopeptidase activity. In human cells, two different ER-resident enzymes, ERAP1 and ERAP2, can trim the N-terminally extended residues of peptide precursors. In this study, the possible cooperative effect of generating five naturally processed HLA-B27 ligands by both proteases was analyzed. We identified differences in the products obtained with increased detection of natural HLA-B27 ligands by comparing double versus single enzyme digestions by mass spectrometry analysis. These in vitro data suggest that each enzyme can use the degradation products of the other as a substrate for new N-terminal trimming, indicating concerted aminoproteolytic activity of ERAP 1 and ERAP2.     

Monoclonal antibodies against an HLA-B27-derived peptide react with an epitope present on bacterial proteins.

The role of molecular mimicry in the spondyloarthropathies was investigated with respect to the epitopes involved. mAb were produced against a synthetic peptide whose sequence was derived from a polymorphic region of the HLA-B27 molecule (amino acids 63-83). Two antibodies (J7F2 and H2B6) were selected for study on the basis of their ability to react with bacterial envelope proteins (ELISA) and B27-positive cells (immunofluorescence). J7F2 reacted preferentially with B27-positive cells and neither antibody reacted with MHC class I negative cells. Based on SDS-PAGE blot analysis of bacterial envelope proteins, the pattern of reactivity for both antibodies (against 36- and 19-kDa proteins) was the same as that for a third monoclonal produced against bacterial envelope and reactive with B27-positive cells. This apparent epitope similarity was investigated by using synthetic peptides to inhibit binding of the monoclonals. The B27 synthetic peptide and a smaller peptide derived from it were efficient inhibitors of antipeptide and antibacterial antibody binding to bacterial Ag and B27-positive cells. These studies provide insight into the molecular basis of cross-reactivity between bacterial proteins and MHC class I molecules.     

Cutting edge: class I presentation of host peptides following HIV infection

Class I MHC molecules bind intracellular peptides for presentation to cytotoxic T lymphocytes. Identification of peptides presented by class I molecules during infection is therefore a priority for detecting and targeting intracellular pathogens. To understand which host-encoded peptides distinguish HIV-infected cells, we have developed a mass spectrometric approach to characterize HLA-B*0702 peptides unique to or up-regulated on infected T cells. In this study, we identify 15 host proteins that are differentially presented on infected human T cells. Peptides with increased expression on HIV-infected cells were derived from multiple categories of cellular proteins including RNA binding proteins and cell cycle regulatory proteins. Therefore, comprehensive analysis of the B*0702 peptide repertoire demonstrates that marked differences in host protein presentation occur after HIV infection.     

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.     

Proteasome-independent HLA-B27 ligands arise mainly from small basic proteins

Many of the constitutive peptide ligands of HLA-B27, a molecule strongly associated with spondyloarthritis, are proteasome-independent. Stable isotope tagging, mass spectrometry, and epoxomicin-mediated inhibition were used to determine their percentage, structural features, and parental proteins. Of 104 molecular species examined, 29.8% were proteasome-independent, paralleling the level of HLA-B27 re-expression in the presence of epoxomicin after acid stripping. Proteasome-dependent and -independent ligands differed little in peptide motifs, flanking sequences, and cellular localization of the parental proteins. In contrast, whereas the former set arose from proteins whose size and isoelectric point distribution largely reflected those in the human proteome, proteasome-independent ligands, other than a few matching signal sequences, were almost totally derived from small (about 6-16.5 kDa) and basic proteins, which account for only 6.6% of the human proteome. Thus, a non-proteasomal proteolytic pathway with strong preference for small proteins is responsible for a significant fraction of the HLA-B27-bound peptide repertoire.     

Computational characterization of residue couplings and micropolymorphism-induced changes in the dynamics of two differentially disease-associated human MHC class-I alleles

Human major histocompatibility complex class I (MHC I) – or human leukocyte antigen (HLA) – proteins present intracellularly processed peptides to cytotoxic T lymphocytes in the adaptive immune response to pathogens. A high level of polymorphism in human MHC I proteins defines the peptide-binding specificity of thousands of different MHC alleles. However, polymorphism as well as the peptide ligand can also affect the global dynamics of the complex. In this study, we conducted classical molecular dynamics simulations of two HLA alleles, the ankylosing spondylitis (AS) associated/tapasin-dependent HLA-B*27:05 and nondisease-associated/tapasin-independent HLA-B*27:09, both in peptide-free forms as well as complex with four different peptides ligands. Our results indicate that in peptide-free form, the single amino acid substitution distinguishing the two alleles (D116H), leads to a weaker dynamic coupling of residues in the tapasin-dependent HLA-B*27:05. In peptide-bound form, several residues of the binding-groove, mostly in A and B pockets, show hinge-like behavior in the global motion of the MHC. Moreover, allele-dependent changes are shown in residue interactions, affecting the B-pocket as well as the beta-2-microglobulin (β2m)-facing residues of the HLA chain.     

Peptide binding to HLA-A2 and HLA-B27 isolated from Escherichia coli. Reconstitution of HLA-A2 and HLA-B27 heavy chain/beta 2-microglobulin complexes requires specific peptides.

The specificity of peptide binding by human leukocyte antigen (HLA) class I molecules was investigated in a cell-free direct-binding assay. Peptides were assessed for binding to HLA-A2 and HLA-B27 by measuring the formation of heterotrimeric HLA complexes that consisted of iodinated beta 2-microglobulin, HLA heavy chain fragments isolated from the Escherichia coli cytoplasm, and peptide. In this system, no detectable HLA heavy chain-beta 2-microglobulin complexes were formed unless appropriate peptides were intentionally added to the reconstitution solution. Analysis with monoclonal antibodies demonstrated that these heterotrimeric complexes were correctly folded. Five nonhomologous peptides, known to form complexes with HLA-A2 or HLA-B27 from T-cell functional studies, were tested for their capacity to bind to HLA-A2 and HLA-B27 using the reconstitution assay. Four of the peptides bound to the appropriate class I molecule only. One peptide and some (but not all) substitution analogs of it bound to both HLA-A2 and HLA-B27. The effect of peptide length on binding to HLA-B27 was studied, and it was found that the optimal length was 9 or 10 amino acid residues; however, one peptide that bound to HLA-B27 was 15 amino acids long. All peptides that bound to HLA-B27 in the direct-binding assay also competed with antigenic peptides for binding to HLA-B27 on the surface of intact cells, as determined by a standard cytotoxic T-lymphocyte functional assay. Thus, we conclude that HLA-A2 and HLA-B27 bind distinct but partially overlapping sets of peptides and that, at least in vitro, the assembly of HLA heavy chain-beta 2-microglobulin complexes requires specific peptides.     

Qualitative and quantitative differences in peptides bound to HLA-B27 in the presence of mouse versus human tapasin define a role for tapasin as a size-dependent peptide editor

Tapasin (Tpn) is a chaperone of the endoplasmic reticulum involved in peptide loading to MHC class I proteins. The influence of mouse Tpn (mTpn) on the HLA-B*2705-bound peptide repertoire was analyzed to characterize the species specificity of this chaperone. B*2705 was expressed on Tpn-deficient human 721.220 cells cotransfected with human (hTpn) or mTpn. The heterodimer to beta(2)-microglobulin-free H chain ratio on the cell surface was reduced with mTpn, suggesting lower B*2705 stability. The B*2705-bound peptide repertoires loaded with hTpn or mTpn shared 94-97% identity, although significant differences in peptide amount were observed in 16-17% of the shared ligands. About 3-6% of peptides were bound only with either hTpn or mTpn. Nonamers differentially bound with mTpn had less suitable anchor residues and bound B*2705 less efficiently in vitro than those loaded only with hTpn or shared nonamers. Decamers showed a different pattern: those found only with mTpn had similarly suitable residues as shared decamers and bound B*2705 with high efficiency. Peptides differentially presented by B*2705 on human or mouse cells showed an analogous pattern of residue suitability, suggesting that the effect of mTpn on B*2705 loading is comparable in both cell types. Thus, mTpn has quantitative and qualitative effects on the B*2705-bound peptide repertoire, impairing presentation of some suitable ligands and allowing others with suboptimal anchor residues and lower affinity to be presented. Our results favor a size-dependent peptide editing role of Tpn for HLA-B*2705 that is species-dependent and suboptimally performed, at least for nonamers, by mTpn.     

A human CTL recognizes a caspase-8-derived peptide on autologous HLA-B*3503 molecules and two unrelated peptides on allogeneic HLA-B*3501 molecules

A CTL clone that recognizes autologous tumor cells was previously isolated from the blood of a head-and-neck cancer patient. The Ag was identified as peptide FPSDSWCYF presented by autologous HLA-B*3503 molecules. This peptide was encoded by a mutated CASP-8 gene, which is implicated in the triggering of apoptosis. Here, we show that this CTL clone, which expresses a single TCR, also recognizes two unrelated peptides on allogeneic HLA-B*3501 molecules. One peptide, HIPDVITY, is encoded by squalene synthase, and the other one, QFADVIVLF, is encoded by 2-hydroxyphytanoyl-CoA lyase. Both genes are expressed ubiquitously. These antigenic peptides are processed and presented by HLA-B*3501 cells. The two HLA-B35 alleles are closely related. Our results might reinforce the notion that the recognition of allogeneic HLA molecules depends on the presence in their groove of a limited number of peptides processed from ubiquitous proteins.     

Gorillas with spondyloarthropathies express an MHC class I molecule with only limited sequence similarity to HLA-B27 that binds peptides with arginine at P2

The human MHC class I gene, HLA-B27, is a strong risk factor for susceptibility to a group of disorders termed spondyloarthropathies (SpAs). HLA-B27-transgenic rodents develop SpAs, implicating HLA-B27 in the etiology of these disorders. Several nonhuman primates, including gorillas, develop signs of SpAs indistinguishable from clinical signs of humans with SpAs. To determine whether SpAs in gorillas have a similar HLA-B27-related etiology, we analyzed the MHC class I molecules expressed in four affected gorillas. Gogo-B01, isolated from three of the animals, has only limited similarity to HLA-B27 at the end of the alpha1 domain. It differs by several residues in the B pocket, including differences at positions 45 and 67. However, the molecular model of Gogo-B*0101 is consistent with a requirement for positively charged residues at the second amino acid of peptides bound by the MHC class I molecule. Indeed, the peptide binding motif and sequence of individual ligands eluted from Gogo-B*0101 demonstrate that, like HLA-B27, this gorilla MHC class I molecule binds peptides with arginine at the second amino acid position of peptides bound by the MHC class I molecule. Furthermore, live cell binding assays show that Gogo-B*0101 can bind HLA-B27 ligands. Therefore, although most gorillas that develop SpAs express an MHC class I molecule with striking differences to HLA-B27, this molecule binds peptides similar to those bound by HLA-B27.     

Targeted Delivery of an Antigenic Peptide to the Endoplasmic Reticulum: Application for Development of a Peptide Therapy for Ankylosing Spondylitis

The development of suitable methods to deliver peptides specifically to the endoplasmic reticulum (ER) can provide some potential therapeutic applications of such peptides. Ankylosing spondylitis (AS) is strongly associated with the expression of human leukocytic antigen-B27 (HLA-B27). HLA-B27 heavy chain (HC) has a propensity to fold slowly resulting in the accumulation of misfolded HLA-B27 HC in the ER, triggering the unfolded protein response, and forming a homodimer, (B27-HC)₂. Natural killer cells and T-helper 17 cells are then activated, contributing to the major pathogenic potentials of AS. The HLA-B27 HC is thus an important target, and delivery of an HLA-B27-binding peptide to the ER capable of promoting HLA-B27 HC folding is a potential mechanism for AS therapy. Here, we demonstrate that a His₆-ubiquitin-tagged Tat-derived peptide (THU) can deliver an HLA-B27-binding peptide to the ER promoting HLA-B27 HC folding. The THU-HLA-B27-binding peptide fusion protein crossed the cell membrane to the cytosol through the Tat-derived peptide. The HLA-B27-binding peptide was specifically cleaved from THU by cytosolic ubiquitin C-terminal hydrolases and subsequently transported into the ER by the transporter associated with antigen processing. This approach has potential application in the development of peptide therapy for AS.     

Toward a definition of self: proteomic evaluation of the class I peptide repertoire

MHC class I molecules present host- and pathogen-derived peptides for immune surveillance. Much attention is given to the search for viral and tumor nonself peptide epitopes, yet the question remains, "What is self?" Analyses of Edman motifs and of small sets of individual peptides suggest that the class I self repertoire consists of thousands of different peptides. However, there exists no systematic characterization of this self-peptide backdrop, causing the definition of class I-presented self to remain largely hypothetical. To better understand the breadth and nature of self proteins sampled by class I HLA, we sequenced >200 endogenously loaded HLA-B*1801 peptides from a human B cell line. Peptide-source proteins, ranging from actin-related protein 6 to zinc finger protein 147, possessed an assortment of biological and molecular functions. Major categories included binding proteins, catalytic proteins, and proteins involved in cell metabolism, growth, and maintenance. Genetically, peptides encoded by all chromosomes were presented. Statistical comparison of proteins presented by class I vs the human proteome provides empiric evidence that the range of proteins sampled by class I is relatively unbiased, with the exception of RNA-binding proteins that are over-represented in the class I peptide repertoire. These data show that, in this cell line, class I-presented self peptides represent a comprehensive and balanced summary of the proteomic content of the cell. Importantly, virus- and tumor-induced changes in virtually any cellular compartment or to any chromosome can be expected to be presented by class I molecules for immune recognition.     

Quantitative and qualitative influences of tapasin on the class I peptide repertoire

Tapasin is critical for efficient loading and surface expression of most HLA class I molecules. The high level surface expression of HLA-B*2705 on tapasin-deficient 721.220 cells allowed the influence of this chaperone on peptide repertoire to be examined. Comparison of peptides bound to HLA-B*2705 expressed on tapasin-deficient and -proficient cells by mass spectrometry revealed an overall reduction in the recovery of B*2705-bound peptides isolated from tapasin-deficient cells despite similar yields of B27 heavy chain and beta(2)-microglobulin. This indicated that a proportion of suboptimal ligands were associated with B27, and they were lost during the purification process. Notwithstanding this failure to recover these suboptimal peptides, there was substantial overlap in the repertoire and biochemical properties of peptides recovered from B27 complexes derived from tapasin-positive and -negative cells. Although many peptides were preferentially or uniquely isolated from B*2705 in tapasin-positive cells, a number of species were preferentially recovered in the absence of tapasin, and some of these peptide ligands have been sequenced. In general, these ligands did not exhibit exceptional binding affinity, and we invoke an argument based on lumenal availability and affinity to explain their tapasin independence. The differential display of peptides in tapasin-negative and -positive cells was also apparent in the reactivity of peptide-sensitive alloreactive CTL raised against tapasin-positive and -negative targets, demonstrating the functional relevance of the biochemical observation of changes in peptide repertoire in the tapasin-deficient APC. Overall, the data reveal that tapasin quantitatively and qualitatively influences ligand selection by class I molecules.     

Conservation and alteration of HLA-B27-specific T cell epitopes on mouse cells. Implications for peptide-mediated alloreactivity.

Alloreactive CTL responses generate a great variety of clonal specificities. Such diversity may be related to recognition of multiple peptides constitutively bound to any given MHC alloantigen. Among human alloreactive CTL, only a fraction of the clones lyse mouse P815 cells expressing class I HLA proteins. In this study the fine specificity of HLA-B27 allorecognition on human or mouse cells by five human HLA-B27-specific CTL clones was comparatively analyzed. This was done to examine what degree of variation in epitope structure is compatible with recognition of HLA Ag on mouse cells. Nine site-specific HLA-B27 mutants were expressed on both human and mouse cells, after DNA-mediated gene transfer, to construct two analogous series of target cells. The reaction patterns of four of the five CTL clones with these cell panels were compatible with conservation of their corresponding epitopes upon expression of HLA-B27 on mouse cells. The reaction pattern of the fifth clone was different with either cell panel, indicating that its epitope was structurally altered on mouse cells. It also suggested a selectively increased expression of the determinant on these cells. The results suggest that most of the epitopes recognized by allospecific CTL clones reacting across species are either independent of any bound peptide or involve identical peptides from both cell types. However, some of these clones recognize alloantigen-bound peptides that are somewhat different in structure depending on the cell type, and may be expressed at the mouse cell surface in greater amounts. Such peptides could arise from related proteins in both species, and be polymorphic as a result of phylogenetic divergence.