Structure-based design of nonnatural ligands for the HLA-B27 protein

X-ray studies as well as structure-activity relationships indicate that the central part of class I MHC-binding nonapeptides represents the main interaction site for a T cell receptor. In order to rationally manipulate T cell epitopes, several nonpeptidic spacer have been designed from the X-ray structure of a MHC-peptide complex and substituted for the T cell receptor-binding part of several antigenic peptides. The binding of the modified epitopes to the HLA-B*2705 protein was studied by an in vitro stabilisation assay and the thermal stability of all complexes examined by circular dichroism spectroscopy. Depending on their chemical nature and length, the introduced spacers may be classified into two categories. Monofunctional spacers (11-amino undecanoate, (R)-3-hydroxybutyrate trimer) simply link two anchoring peptide positions (P3 and P9) but loosely contact the MHC binding groove, and thus decrease more or less the affinity of the altered epitopes to HLA-B*2705. Bifunctional spacers ((R)-3-hydroxybutyrate and beta-homoalanine combinations) not only bridges the two distant anchoring amino acids but also strongly interact with the binding cleft and lead to an increase in binding to the MHC protein. The presented modified ligands constitute interesting tools for perturbing the T cell response to the parent antigenic peptide.     

Conformational dimorphism of self-peptides and molecular mimicry in a disease-associated HLA-B27 subtype

An interesting property of certain peptides presented by major histocompatibility complex (MHC) molecules is their acquisition of a dual binding mode within the peptide binding groove. Using x-ray crystallography at 1.4 A resolution, we show here that the glucagon receptor-derived self-peptide pGR ((412)RRRWHRWRL(420)) is presented by the disease-associated human MHC class I subtype HLA-B*2705 in a dual conformation as well, with the middle of the peptide bent toward the floor of the peptide binding groove of the molecule in both binding modes. The conformations of pGR are compared here with those of another self-peptide (pVIPR, RRKWRRWHL) that is also displayed in two binding modes by HLA-B*2705 antigens and with that of the viral peptide pLMP2 (RRRWRRLTV). Conserved structural features suggest that the N-terminal halves of the peptides are crucial in allowing cytotoxic T lymphocyte (CTL) cross-reactivity. In addition, an analysis of T cell receptors (TCRs) from pGR- or pVIPR-directed, HLA-B27-restricted CTL clones demonstrates that TCR from distinct clones but with comparable reactivity may share CDR3alpha but not CDR3beta regions. Therefore, the cross-reactivity of these CTLs depends on TCR-CDR3alpha, is modulated by TCR-CDR3beta sequences, and is ultimately a consequence of the conformational dimorphism that characterizes binding of the self-peptides to HLA-B*2705. These results lend support to the concept that conformational dimorphisms of MHC class I-bound peptides might be connected with the occurrence of self-reactive CTL.     

Influence of inflammation-related changes on conformational characteristics of HLA-B27 subtypes as detected by IR spectroscopy

Inflammatory processes are accompanied by the post-translational modification of certain arginine residues to yield citrulline, and a pH decrease in the affected tissue, which might influence the protonation of histidine residues within proteins. We employed isotope-edited IR spectroscopy to investigate whether conformational features of two human major histocompatibility antigen class I subtypes, HLA-B*2705 and HLA-B*2709, are affected by these changes. Both differ only in residue 116 (Asp vs. His) within the peptide-binding grooves, but are differentially associated with inflammatory rheumatic disorders. Our analyses of the two HLA-B27 subtypes in complex with a modified self-peptide containing a citrulline RRKWURWHL (U = citrulline) revealed that the heavy chain is more flexible in the HLA-B*2705 subtype than in the HLA-B*2709 subtype. Together with our previous studies of HLA-B27 subtypes complexed with the unmodified self-peptide RRKWRRWHL, these findings support the existence of subtype-specific conformational features of the heavy chains under physiological conditions, which are undetectable by X-ray crystallography and exist irrespective of the sequence of the bound peptide and its binding mode. They might thus influence antigenic properties of the respective HLA-B27 subtype. Furthermore, a decrease in the pH from 7.5 to 5.6 during the analyses had an influence only on HLA-B*2709 complexed with the unmodified self-peptide, where His116 is not contacted by any peptide side chain. This permits us to conclude that histidines, and in particular His116, influence the stability of MHC:peptide complexes. The conditions prevailing in inflammatory environments in vivo might thus also exert an impact on selected conformational features of HLA-B27:peptide complexes.     

Thermodynamic stability of HLA-B*2705. Peptide complexes. Effect of peptide and major histocompatibility complex protein mutations

Designing synthetic vaccines from class I major histocompatibility complex (MHC)-binding antigenic peptides requires not only knowledge of the binding affinity of the designed peptide but also predicting the stability of the formed MHC-peptide complex. In order to better investigate structure-stability relationships, we have determined by circular dichroism spectroscopy the thermal stability of a class I MHC protein, HLA-B*2705, in complex with a set of 39 singly substituted peptide analogues. The influence of two anchoring side chains (P3 and P9) was studied by peptide mutation and appropriate site-directed mutagenesis of the HLA-B*2705 binding groove. The side chain at P9 is clearly the one that contributes the most to the thermal stability of the MHC-peptide complexes, as destabilization up to 25 degrees C are obtained after P9 mutation. Interestingly, structure-stability relationships do not fully mirror structure-binding relationships. As important as the C-terminal side chain are the terminal ammonium and carboxylate groups. Removal of a single H-bond between HLA-B27 and the terminal peptide moieties results in thermal destabilization up to 10 degrees C. Depending on the bound peptide and the location of the deleted H-bond, the decrease in the thermal stability of the corresponding complex is quantitatively different. The present study suggests that any peptidic amino acid at positions 3 and 9 promotes refolding of the B27-peptide complex. Once the complex is formed, the C-terminal side chain seems to play an important role for maintaining a stable complex.     

The solvent-inaccessible Cys67 residue of HLA-B27 contributes to T cell recognition of HLA-B27/peptide complexes

Crystallographic studies have suggested that the cysteine at position 67 (Cys(67)) in the B pocket of the MHC molecule HLA-B*2705 is of importance for peptide binding, and biophysical studies have documented altered thermodynamic stability of the molecule when Cys(67) was mutated to serine (Ser(67)). In this study, we used HLA-B27.Cys(67) and HLA-B27.Ser(67) tetramers with defined T cell epitopes to determine the contribution of this polymorphic, solvent-inaccessible MHC residue to T cell recognition. We generated these HLA-B27 tetramers using immunodominant viral peptides with high binding affinity to HLA-B27 and cartilage-derived peptides with lower affinity. We demonstrate that the yield of refolding of HLA-B27.Ser(67) molecules was higher than for HLA-B27.Cys(67) molecules and strongly dependent on the affinity of the peptide. T cell recognition did not differ between HLA-B27.Cys(67) and HLA.B27.Ser(67) tetramers for the viral peptides that were investigated. However, an aggrecan peptide-specific T cell line derived from an HLA-B27 transgenic BALB/c mouse bound significantly stronger to the HLA-B27.Cys(67) tetramer than to the HLA-B27.Ser(67) tetramer. Modeling studies of the molecular structure suggest the loss of a SH ... pi hydrogen bond with the Cys-->Ser substitution in the HLA-B27 H chain which reduces the stability of the HLA-B27/peptide complex. These results demonstrate that a solvent-inaccessible residue in the B pocket of HLA-B27 can affect TCR binding in a peptide-dependent fashion.     

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     

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.     

Allospecific T cell epitope sharing reveals extensive conservation of the antigenic features of peptide ligands among HLA-B27 subtypes differentially associated with spondyloarthritis

HLA-B*2702, B*2704, and B*2705 are strongly associated with spondyloarthritis, whereas B*2706 is not. Subtypes differ among each other by a few amino acid changes and bind overlapping peptide repertoires. In this study we asked whether differential subtype association with disease is related to differentially bound peptides or to altered antigenicity of shared ligands. Alloreactive CTL raised against B*2704 were analyzed for cross-reaction with B*2705, B*2702, B*2706, and mutants mimicking subtype changes. These CTL are directed against many alloantigen-bound peptides and can be used to analyze the antigenicity of HLA-B27 ligands on different subtypes. Cross-reaction of anti-B*2704 CTL with B*2705 and B*2702 correlated with overlap of their peptidic anchor motifs, suggesting that many shared ligands have similar antigenic features on these three subtypes. Moreover, the percent of anti-B*2704 CTL cross-reacting with B*2706 was only slightly lower than the overlap between the corresponding peptide repertoires, suggesting that most shared ligands have similar antigenic features on these two subtypes. Cross-reaction with B*2705 or mutants mimicking changes between B*2704 and B*2705 was donor-dependent. In contrast, cross-reaction with B*2702 or B*2706 was less variable among individuals. Conservation of antigenic properties among subtypes has implications for allorecognition, as it suggests that shared peptides may determine cross-reaction across exposed amino acid differences in the MHC molecules and that the antigenic distinctness of closely related allotypes may differ among donors. Our results also suggest that differential association of HLA-B27 subtypes with spondyloarthritis is more likely related to differentially bound peptides than to altered antigenicity of shared ligands.     

Two HLA-B14 subtypes (B*1402 and B*1403) differentially associated with ankylosing spondylitis differ substantially in peptide specificity but have limited peptide and T-cell epitope sharing with HLA-B27

The peptide specificity of HLA-B*1403, an allotype associated with ankylosing spondylitis (Lopez-Larrea, C., Mijiyawa, M., Gonzalez, S., Fernandez-Morera, J. L., Blanco-Gelaz, M. A., Martinez-Borra, J., and Lopez-Vazquez, A. (2002) Arthritis Rheum. 46, 2968-2971) was compared with those of the non-associated B*1402 and the prototypic disease-associated B*2705 allotypes. Although differing by a single residue (L156R), B*1402 and B*1403 shared only 32-35% of their peptide repertoires. Subtype-related differences observed in multiple peptide positions, including P3 and P7, were largely explained by a direct effect of the L156R change on peptide specificity. The HLA-B14 subtypes shared only approximately 3% of their peptide repertoires with B*2705. This was due to distinct residue usage at most positions, as revealed by statistical comparison of B*1402, B*1403, and B*2705-bound nonamers. Nevertheless, shared ligands between B*2705 and B*1403 were formally identified, although ligands common to B*2705 and B*1403, but absent from B*1402, were not found. Alloreactive T-cells were used as a tool to analyze epitope sharing among B*1402, B*1403, and B*2705. The percentage of cross-reactive T-cell clones closely paralleled peptide overlap, suggesting that shared ligands tend to maintain their antigenic features when bound to the different allotypes. Our results indicate that B*1403 and B*2705 can present common peptides. However, both the disparity of their peptide repertoires and the lack of binding features shared by these two allotypes, but not B*1402, argue against, although do not exclude, a mechanism of spondyloarthritis mediated by specific ligands of B*2705 and B*1403.     

High T cell epitope sharing between two HLA-B27 subtypes (B*2705 and B*2709) differentially associated to ankylosing spondylitis.

HLA-B*2705 is strongly associated with ankylosing spondylitis (AS) and reactive arthritis. In contrast, B*2709 has been reported to be more weakly or not associated to AS. These two molecules differ by a single amino acid change: aspartic acid in B*2705 or histidine in B*2709 at position 116. In this study, we analyzed the degree of T cell epitope sharing between the two subtypes. Ten allospecific T cell clones raised against B*2705, 10 clones raised against B*2703 but cross-reactive with B*2705, and 10 clones raised against B*2709 were examined for their capacity to lyse B*2705 and B*2709 target cells. The anti-B*2705 and anti-B*2703 CTL were peptide dependent as demonstrated by their failure to lyse TAP-deficient B*2705-T2 transfectant cells. Eight of the anti-B*2705 and five of the anti-B*2703 CTL clones lysed B*2709 targets. The degree of cross-reaction between B*2705 and B*2709 was donor dependent. In addition, the effect of the B*2709 mutation (D116H) on allorecognition was smaller than the effect of the other naturally occurring subtype change at this position, D116Y. These results demonstrate that B*2705 and B*2709 are the antigenically closest HLA-B27 subtypes. Because allospecific T cell recognition is peptide dependent, our results imply that the B*2705- and B*2709-bound peptide repertoires are largely overlapping. Thus, to the extent to which linkage of HLA-B27 with AS is related to the peptide-presenting properties of this molecule, our results would imply that peptides within a relatively small fraction of the HLA-B27-bound peptide repertoire influence susceptibility to this disease.     

Peptide-presenting similarities among functionally distant HLA-B27 subtypes revealed by alloreactive T lymphocytes of unusual specificity.

Functional dissection of HLA-B27 subtypes using alloreactive or B27-restricted CTL has shown that the structurally related B*2704 and B*2706 are the most distant subtypes relative to the prototype B*2705. In particular, previous studies have failed to find anti-B*2705 CTL cross-reacting with B*2704 or B*2706. Such failure can be accounted for by the drastic effect on T cell recognition of the change at residue 152 in both subtypes relative to B*2705, as established with site-directed mutants. B*2704 and B*2706 are also related in ethnic distribution, as they are restricted to Orientals, jointly being the predominant HLA-B27 subtypes in this population. As far as it is known, there are no differences relative to B*2705 in their linkage to ankylosing spondylitis. In our study, 5 of 13 examined anti-B*2705 limiting dilution CTL lines from a particular HLA-B27- individual were shown to crossreact with B*2704, B*2706 or both. The monoclonal nature of this cross-reaction was established by cold target competition analysis. This result demonstrates that the apparent differences in T cell antigenicity among anti-B27 subtypes are strongly influenced by the responder individual, as the spectrum of clonal specificities in anti-B27 responses may show significant differences among unrelated responders. Fine specificity differences among the cross-reactive CTL allowed unambiguous functional distinction between B*2704 and B*2706. The molecular basis of such cross-reactivity was examined by correlating CTL reaction patterns with the structure of both subtypes, which differ only by two residues located in the beta-pleated sheet bottom of the peptide binding site, and with site-directed mutants mimicking HLA-B27 subtype polymorphism. The results suggest that: 1) distinct peptides are involved in the allospecific epitopes recognized by the various crossreactive CTL, and 2) B*2704, B*2706, and B*2705 differ in their peptide-presenting specificity, but can present some identical or structurally similar peptides.     

Binding of rationally designed non-natural peptides to the human leukocyte antigen HLA-B*2705

High-affinity ligands of non-peptidic nature, binding to the class I major histocompatibility complex protein HLA B*2705 whose expression is strongly linked to the pathogenesis of the autoimmune disease ankylosing spondylitis, should give way to a selective immunotherapy by blocking or antagonising the interaction with autoreactive T cell clones. Here we present experimental data on the binding of modified peptides, designed to optimally bind to HLA-B*2705 by filling a hydrophobic binding pocket (pocket D) with nonencoded aromatic amino acids. Three peptides with altered side chains (alpha-naphthylalanine, beta-naphthylalanine and homophenylalanine) in position 3 were synthesised. The thermal denaturation profiles of the HLA protein in complex with the modified peptides, monitored by circular dichroism spectroscopy, showed a significant shift towards higher melting temperatures with respect to the parent T cell epitope. The proposed binding mode of the nonnatural peptides was checked by site-directed mutagenesis of the pocket D, hypothesised to accommodate the large hydrophobic side chains. Reducing the size and depth of the pocket by mutating Leu l56 into Trp only affects the binding of the non-natural ligands, thus providing experimental evidence that the nonnatural peptide amino acids bind as predicted to the host MHC protein. © 1998 European Peptide Society and John Wiley & Sons, Ltd.     

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.     

Differential peptide dynamics is linked to major histocompatibility complex polymorphism

Peptide presentation by major histocompatibility complex (MHC) molecules is of central importance for immune responses, which are triggered through recognition of peptide-loaded MHC molecules (pMHC) by cellular ligands such as T-cell receptors (TCR). However, a unifying link between structural features of pMHC and cellular responses has not been established. Instead, pMHC/TCR binding studies suggest conformational and/or flexibility changes of the binding partners as a possible cause of differential T-cell stimulation, but information on real-time dynamics is lacking. We therefore probed the real-time dynamics of a MHC-bound nonapeptide (m9), by combining time-resolved fluorescence depolarization and molecular dynamics simulations. Here we show that the nanosecond dynamics of this peptide presented by two human MHC class I subtypes (HLA-B*2705 and HLA-B*2709) with differential autoimmune disease association varies dramatically, despite virtually identical crystal structures. The peptide dynamics is linked to the single, buried polymorphic residue 116 in the peptide binding groove. Pronounced peptide flexibility is seen only for the non-disease-associated subtype HLA-B*2709, suggesting an entropic control of peptide recognition. Thermodynamic data obtained for two additional peptides support this hypothesis.     

Assembly and intracellular trafficking of HLA-B*3501 and HLA-B*3503

Residue 116 of major histocompatibility complex (MHC) class I heavy chains is an important determinant of assembly, that can influence rates of ER-Golgi trafficking, binding to the transporter associated with antigen processing (TAP), tapasin dependence of assembly, and the efficiency and specificity of peptide binding. Here, we investigated assembly and peptide-binding differences between HLA-B*3501(S116) and HLA-B*3503(F116), two alleles differing only at position 116 of the MHC class I heavy chain, that are associated respectively with normal or rapid AIDS progression. A reduced intracellular maturation rate was observed for HLA-B*3503 in HIV-infected and uninfected cells, which correlated with enhanced binding of HLA-B*3503 to TAP. No significant differences in the intrinsic efficiency of in vitro peptide binding by HLA-B*3501 and HLA-B*3503 were measurable with several common peptides or peptide libraries, and both allotypes were relatively tapasin-independent for their assembly. However, thermostability differences between the two allotypes were measurable in a CD4⁺ T cell line. These findings suggest that compared to HLA-B*3501, a reduced intracellular peptide repertoire for HLA-B*3503 could contribute to its slower intracellular trafficking and stronger association with rapid AIDS progression.     

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).     

Preferential HLA usage in the influenza virus-specific CTL response

To study whether individual HLA class I alleles are used preferentially or equally in human virus-specific CTL responses, the contribution of individual HLA-A and -B alleles to the human influenza virus-specific CTL response was investigated. To this end, PBMC were obtained from three groups of HLA-A and -B identical blood donors and stimulated with influenza virus. In the virus-specific CD8(+) T cell population, the proportion of IFN-gamma- and TNF-alpha-producing cells, restricted by individual HLA-A and -B alleles, was determined using virus-infected C1R cells expressing a single HLA-A or -B allele for restimulation of these cells. In HLA-B*2705- and HLA-B*3501-positive individuals, these alleles were preferentially used in the influenza A virus-specific CTL response, while the contribution of HLA-B*0801 and HLA-A*0101 was minor in these donors. The magnitude of the HLA-B*0801-restricted response was even lower in the presence of HLA-B*2705. C1R cells expressing HLA-B*2705, HLA-A*0101, or HLA-A*0201 were preferentially lysed by virus-specific CD8(+) T cells. In contrast, the CTL response to influenza B virus was mainly directed toward HLA-B*0801-restricted epitopes. Thus, the preferential use of HLA alleles depended on the virus studied.     

Molecular determinants of major histocompatibility complex class I complex stability: shaping antigenic features through short and long range electrostatic interactions

A single amino acid exchange between the major histocompatibility complex molecules HLA-B(*)2705 and HLA-B(*)2709 (Asp-116/His) is responsible for the emergence of distinct HLA-B27-restricted T cell repertoires in individuals harboring either of these two subtypes and could correlate with their differential association with the autoimmune disease ankylosing spondylitis. By using fluorescence depolarization and pK(a) calculations, we investigated to what extent electrostatic interactions contribute to shape antigenic differences between these HLA molecules complexed with viral, self, and non-natural peptide ligands. In addition to the established main anchor of peptides binding to HLA-B27, arginine at position 2 (pArg-2), and the secondary anchors at the peptide termini, at least two further determinants contribute to stable peptide accommodation. 1) The interaction of Asp-116 with arginine at peptide position 5, as found in pLMP2 (RRRWRRLTV; viral) and pVIPR (RRKWRRWHL; self), and with lysine in pOmega, as found in gag (KRWIILGLNK; viral), additionally stabilizes the B(*)2705 complexes by approximately 5 and approximately 27 kJ/mol, respectively, in comparison with B(*)2709. 2) The protonation state of the key residues Glu-45 and Glu-63 in the B-pocket, which accommodates pArg-2, affects peptide binding strength in a peptide- and subtype-dependent manner. In B(*)2705/pLMP2, protonation of Glu-45/Glu-63 reduces the interaction energy of pArg-2 by approximately 24 kJ/mol as compared with B(*)2705/pVIPR. B(*)2705/pVIPR is stabilized by a deprotonated Glu-45/Glu-63 pair, evoked by allosteric interactions with pHis-8. The mutual electrostatic interactions of peptide and HLA molecule, including peptide- and subtype-dependent protonation of key residues, modulate complex stability and antigenic features of the respective HLA-B27 subtype.     

C-terminal anchoring of a peptide to class II MHC via the P10 residue is compatible with a peptide bulge.

The binding of antigenic peptide to class II MHC is mediated by hydrogen bonds between the MHC and the peptide, by salt bridges, and by hydrophobic interactions. The latter are confined to a number of deeper pockets within the peptide binding groove, and peptide side chains that interact with these pockets are referred to as anchor residues. T cell recognition involves solvent-accessible peptide residues along with minor changes in MHC helical pitch induced by the anchor residues. In class I MHC there is an added level of epitope complexity that results from binding of longer peptides that bulge out into the solvent-accessible, T cell contact area. Unlike class I MHC, class II MHC does not bind peptides of discrete length, and the possibility of peptide bulging has not been clearly addressed. A peptide derived from position 24-37 of integrin beta(3) can either bind or not bind to the class II MHC molecule HLA DRB3*0101 based on a polymorphism at the P9 anchor. We show that the loss of binding can be compensated by changes at the P10 position. We propose that this could be an example of a class II peptide bulge. Although not as efficient as P9 anchoring, the use of P10 as an anchor adds another possible mechanism by which T cell epitopes can be generated in the class II presentation system.     

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.