Alloreactive cytolytic T cell clones with dual recognition of HLA-B27 and HLA-DR2 antigens. Selective involvement of CD8 in their class I--directed cytotoxicity.

HLA-B27- responder cells were stimulated in vitro with HLA-B27.1+ lymphoblastoid cell lines, and alloreactive CTL clones were obtained by limiting dilution. Three of these clones specifically lysed B27.1+ targets. In addition, they also lysed homozygous DR2 targets with various degrees of efficiency, depending on the Dw specificity of the target cell. All three clones possessed a homogeneous CD3+,CD8+,CD4- phenotype and were also homogeneous upon subcloning. Cold-target inhibition analyses showed mutual inhibition of B27.1 target lysis by DR2 targets and vice versa. Lysis of B27.1 targets was selectively inhibited by anti-class I mAbs. In contrast, lysis of DR2 targets was inhibited only by anti-class II and anti-DR monomorphic antibodies, but not by anti-class I, anti-DQw1, or anti-DP antibodies. The results indicate that these clones display dual recognition for HLA-B27.1 and for HLA-DR2 and suggest that HLA-B27.1 may share at least one epitope that is closely related to some stimulatory Dw determinants present on the HLA-DR2 antigens. Lysis of both B27+ and DR+ targets was inhibited by an anti-CD3 mAb. In contrast, an anti-CD8 antibody selectively inhibited the B27- but not the DR2-directed killing by these clones. The data support a stabilizing role of CD8 through its binding to the same class I (but not class II) molecule on the target cell bound by the T cell antigen receptor.

T cell receptor V beta gene usage in a human alloreactive response. Shared structural features among HLA-B27-specific T cell clones.

A strategy, based on using V beta family-specific oligonucleotides, was developed for specific amplification and direct sequencing of human TCR V beta genes. With this strategy, it was possible to undertake a structural analysis of TCRs from human T cell clones in specific responses. 12 HLA-B27-specific cytotoxic clones were examined. The results reveal a nonrandom use of V beta gene diversity in this alloreactive response in that: (a) the clones express a restricted number of V beta segments, including a subset of V beta families that are significantly more related to one another than to most other V beta families; (b) five of seven clones having a particular reaction pattern with HLA-B27 subtypes possess Alanine at the D-J junction; and (c) identical J beta segments are found associated in several instances with identical or highly homologous V beta gene segments. In addition, two new V beta 13 members are reported.

Changes in the repertoire of peptides bound to HLA-B27 subtypes and to site-specific mutants inside and outside pocket B.

HLA-B27 subtypes share many structural features, including their pocket B, which interacts with a conserved Arg residue at the second position of B*2705-bound peptides. Subtypes differ among each other at other locations in the peptide binding site. In this study, metabolic labeling and radiochemical pool sequencing were used to address the following issues: (a) presence of the Arg 2 (R2) motif among peptides bound to the various HLA-B27 subtypes; (b) influence of mutations inside and outside pocket B on this motif; and (c) the degree of similarity among the peptide pools bound to the various B27 subtypes. Sequencing of Arg-labeled peptide pools extracted from B*2701 to B*2706, and from two site-directed mutants of B*2705 with changes outside pocket B, indicated that all of these molecules bind peptides with Arg at position 2. Peptides from several mutants with changes altering the structure of pocket B, and from one mutant at the pocket B rim, also retained the R2 motif. However, this was absent in the peptide pool extracted from the M45 mutant, in which the negative charge of pocket B, conferred to HLA-B27 by Glu45, was canceled. These results indicate that alterations outside pocket B, and even disruption of the network of hydrogen bonds that stabilizes Arg binding in pocket B, do not impair binding of peptides bearing the R2 motif, but a nonconservative substitution at position 45 does. As a substantial fraction of anti-B*2705 cytotoxic T lymphocyte (CTL) clones crossreact with the M45 mutant (Villadangos, J., B. Galocha, D. López, V. Calvo, and J. A. López de Castro. 1992. J. Immunol. 149:505) this result suggest that determinant mimicry by nonidentical peptides may frequently account for unexpected CTL crossreactions. Metabolic labeling with various other amino acids and radiochemical sequencing revealed similarities, but also substantial differences, among the peptide pools from the various HLA-B27 subtypes. This strongly suggests that many peptides bind to multiple subtypes, but significant subsets of peptides bound to a given HLA-B27 subtype do not bind to other subtypes or do so with greatly altered efficiency. These results indicate the importance of polymorphism outside pocket B in modulating peptide binding to HLA-B27.

The pathogenetic role of HLA-B27 in chronic arthritis.

Population and peptide specificity analyses and studies in transgenic rodents support a role of HLA-B27 as an antigen-presenting molecule in spondyloarthropathy. The interplay between HLA-B27 and arthritogenic bacteria on infected cells suggests that HLA-B27 might also influence disease by other mechanisms. Recent genetic advances promise the identification of additional susceptibility genes.

A post-translational modification of nuclear proteins, N(G),N(G)-dimethyl-Arg, found in a natural HLA class I peptide ligand.

Presentation of peptides derived from endogenous proteins by class I major histocompatibility complex molecules is essential both for immunological self-tolerance and induction of cytotoxic T-cell responses against intracellular parasites. Despite frequent and diverse post-translational modification of eukaryotic cell proteins, very few class I-bound peptides with post-translationally modified residues are known. Here we describe a natural dodecamer ligand of HLA-B39 (B*3910) derived from an RNA-binding nucleoprotein that carried N(G),N(G)-dimethyl-Arg. Although common among RNA-binding proteins, this modification was not previously known among natural class I ligands. The sequence of this peptide was determined by Edman degradation and electrospray ion trap mass spectrometry. The fragmentation pattern of the dimethyl-Arg side chain observed with this latter technique allowed us to unambiguously assign the isomeric form of the modified residue. The post-translationally modified ligand was a prominent component (1-2%) of the B*3910-bound peptide repertoire. The dimethyl-Arg residue was located in a central position of the peptide, amenable to interacting with T-cell receptors, and most other residues in the middle region of the peptide were Gly. These structural features strongly suggest that the post-translationally modified residue may have a major influence on the antigenic properties of this natural ligand.

Long-range effects in protein--ligand interactions mediate peptide specificity in the human major histocompatibilty antigen HLA-B27 (B*2701).

B*2701 differs from all other HLA-B27 subtypes of known peptide specificity in that, among its natural peptide ligands, arginine is not the only allowed residue at peptide position 2. Indeed, B*2701 is unique in binding many peptides with Gln2 in vivo. However, the mutation (Asp74Tyr) responsible for altered selectivity is far away from the B pocket of the peptide binding site to which Gln/Arg2 binds. Here, we present a model that explains this effect. It is proposed that a new rotameric state of the conserved Lys70 is responsible for the unique B*2701 binding motif. This side chain should be either kept away from pocket B through its interaction with Asp74 in most HLA-B27 subtypes, or switched to this pocket if residue 74 is Tyr as in B*2701. Involvement of Lys70 in pocket B would thus allow binding of peptides with Gln2. Binding of Arg2-containing peptides to B*2701 is also possible because Lys70 could adopt another conformation, H-bonded to Asn97, which preserves the same binding mode of Arg2 as in B*2705. This model was experimentally validated by mutating Lys70 into Ala in B*2701. Edman sequencing of the B*2701(K70A) peptide pool showed only Arg2, characteristic of HLA-B27-bound peptides, and no evidence for Gln2. This supports the computational model and demonstrates that allowance of B*2701 for peptides with Gln2 is due to the long-range effect of the polymorphic residue 74 of HLA-B27, by inducing a conformational switch of the conserved Lys70.

Nonapeptide analogues containing (R)-3-hydroxybutanoate and beta-homoalanine oligomers: synthesis and binding affinity to a class I major histocompatibility complex protein.

Crystal structures of antigenic peptides bound to class I MHC proteins suggest that chemical modifications of the central part of the bound peptide should not alter binding affinity to the MHC restriction protein but could perturb the T-cell response to the parent epitope. In our effort in designing nonpeptidic high-affinity ligands for class I MHC proteins, oligomers of (R)-3-hydroxybutanoate and(or) beta-homoalanine have been substituted for the central part of a HLA-B27-restricted T-cell epitope of viral origin. The affinity of six modified peptides to the B2705 allele was determined by an in vitro stabilization assay. Four out of the six designed analogues presented an affinity similar to that of the parent peptide. Two compounds, sharing the same stereochemistry (R,R,S,S) at the four stereogenic centers of the nonpeptidic spacer, bound to B2705 with a 5-6-fold decreased affinity. Although the chiral spacers do not strongly interact with the protein active site, there are configurations which are not accepted by the MHC binding groove, probably because of improper orientation of some lateral substituents in the bound state and different conformational behavior in the free state. However we demonstrate that beta-amino acids can be incorporated in the sequence of viral T-cell epitopes without impairing MHC binding. The presented structure-activity relationships open the door to the rational design of peptide-based vaccines and of nonnatural T-cell receptor antagonists aimed at blocking peptide-specific T-cell responses in MHC-associated autoimmune diseases.

The effect of the chemical modification in human Fc gamma fragment on protein A and Fc receptor binding.

The role of acidic side-chains on Fc gamma fragment in granulocyte receptor binding and in S. aureus protein A binding has been investigated by means of chemical modification. Alteration of a restricted number of carboxyl groups after 5 min of reaction is sufficient to abrogate the capacity of Fc to inhibit EA rosette formation by human neutrophils. More limited modification, which affects mainly the most exposed acidic chains, does not change receptor binding activity. In contrast, the interaction with protein A is largely unaffected, even under reaction conditions which are able to induce significant changes in the circular dichroism spectrum of Fc fragment. The results suggest that some acidic groups on Fc may be involved in the interaction with neutrophil receptor and that the binding to protein A and Fc receptor involves different sites.

CD8 is involved in both class I- and class II-induced proliferation of a cytolytic T-cell clone with dual specificity for HLA-B27 and HLA-DR2 antigens.

A CD3+ CD4- CD8+ cytolytic T-lymphocyte (CTL) clone, CTL 47, could be induced to proliferate in the presence of exogenous interleukin 2 by either HLA-B27.1+ or HLA-DR2+ cells. B27.1-induced proliferation was strongly and equally inhibited by an anti-B27 and by an anti-CD8 monoclonal antibody (MoAb). DR2-induced proliferation was inhibited by the same anti-CD8 MoAb less efficiently and with a different time course than anti-class II blocking, only being significant when the antibody was added ab initio or very early during the assay. These results indicate that CD8 is essential for class I-induced proliferation but that it also enhances class II-induced stimulation of this CTL clone. It is proposed that the necessary role of CD8 in class I-induced proliferation is related to its interaction with the same class I molecule bound by the T-cell receptor. The accessory role in class II-induced proliferation would be due to an additive effect on the avidity of cell adhesion, resulting from interaction of CD8 with the class I antigens on the stimulator cell, or perhaps to a regulatory role of CD8 as a transducer of early signals for T-cell activation.

Antigenicity of HLA-A2 and HLA-B7. Loss and gain of serologic determinants induced by site-specific mutagenesis at residues 62 to 80.

The contribution of the hypervariable region spanning amino acid residues 62 to 80 to the serologic determinants of HLA-A2 and HLA-B7 has been examined by site-directed mutagenesis. Three HLA-A2 mutants, having changes as in HLA-B7 at positions 62, 76, and at the complete 65-to-80 segment, respectively, were obtained and expressed on class I HLA-deficient human cells upon transfection. The reactivity of 19 monoclonal antibodies (mAbs) against both broad public and allospecific determinants on HLA-A2 and HLA-B7 was analyzed. The results indicate that: (1) the change at residue 62 abrogated recognition of the corresponding HLA-A2 mutant by mAb MA2.1 (anti-A2 + B17); (2) the change at residue 76 did not effect any of the determinants analyzed, although its side chain is easily accessible at the surface of the molecule; (3) the replacement of the whole 65-to-80 segment in HLA-A2 by that from HLA-B7 abrogated recognition by MA2.1 and by 108-2C5, a mAb recognizing a public determinant from the HLA-A locus. Such replacement led to gaining the determinants recognized by mAbs GS145.2 (anti-B7 + B27) and SFR8-B6 (anti-Bw6); and (4) the HLA-A2-reactive mAbs whose reactivity was known to be abrogated by changes in alpha 2 were unaffected by the changes introduced in alpha 1, underlining the frequent segregation of serologic determinants on class I antigens to single domains.

Conventional alloantisera can recognize the same HLA-B27 polymorphism as detected by cytotoxic T lymphocytes.

Subtypes of B27 have been identified by cytotoxic T lymphocytes, biochemistry, molecular biology, and murine monoclonal antibodies. In the present study we describe seven B27 subtype-specific pregnancy sera. The reaction pattern of these B27 subtype-specific sera closely parallels the recognition pattern of B27 subtype-specific cytotoxic T lymphocytes. Because the complete amino acid sequence of the studied B27 subtypes (B27W, B27K, B27C, B27D) is known, it can be determined which amino acid substitutions are responsible for recognition by subtype-specific sera and by cytotoxic T lymphocytes, respectively. It is proposed that B27 subtype-specific sera and B27 subtype-specific cytotoxic T lymphocytes recognize the same epitopes or that a single amino acid change can induce multiple antigenic determinants, which are recognized differentially by antibodies and T cells.

High-sensitivity analysis and sequencing of peptides and proteins by quadrupole ion trap mass spectrometry.

This paper describes experience with the commercially available LCQ quadrupole ion trap mass spectrometer applied to the off-line analysis of peptides and proteins. The standard front end of the electrospray probe was replaced with a micromanipulator which, with the aid of a magnifying device, allowed the use of a variety of miniaturized spraying interfaces. The low sample consumption and extended analysis times of these devices were ideally suitable to obtain improved results in terms of sensitivity and mass accuracy. This needed a careful optimization of the number of ions stored inside the trap (ion target parameter) and required spectrum averaging of many scans. A method is presented for the mathematical fitting of ZoomScan spectra to theoretical isotopic distributions, which allowed the mass determination of large peptides with more accuracy than that achieved by conventional deconvolution algorithms. A very simple on-line desalting configuration is also described which needed no external micro-high-performance liquid chromatographic pumps, and can be easily mounted using the built-in syringe delivery system of the LCQ. This set-up allowed extended analysis times of 'in-gel' protein digests in subpicomole amounts. Finally, the multiple fragmentation capabilities of the ion trap were found to be extremely useful for the analysis of peptide modifications such as phosphorylation and for sequencing individual peptides from highly complex MHC-bound peptide pools.

HLA-B27 and HLA-B73 polymorphism and its role on antigenicity, peptide presentation, and disease susceptibility.

Current ideas on the structure, evolution, and disease association of HLA-B27 antigens are presented, with emphasis on the effect of HLA-B27 polymorphism on T-cell recognition and antigen-presenting properties of these molecules. The molecular relationship between HLA-B27 and HLA-B73, and its implications for a putative pathogenetic role of the later antigen are discussed.

Peptides: the cornerstone of HLA-B27 biology and pathogenetic role in spondyloarthritis.

The association of human leukocyte antigen (HLA)-B27 to ankylosing spondylitis is one of the strongest between a major histocompatibility complex molecule and a disease. Yet, the basis for this association remains unknown. Several hypotheses, each based on a particular feature of HLA-B27, guide much of the current research on the pathogenesis of this disease, but none has yet satisfactorily explained its mechanism and the differential association of B27 subtypes to it. In this review, the pathogenetic role of HLA-B27 will be analyzed from a global perspective of its biology, emphasizing the interdependency of multiple molecular features and the likely influence of disease-modifying gene products. From this perspective, peptide binding emerges as the cornerstone of all other biological properties.

HLA-B27 and HLA-A2 subtypes: structure, evolution and function.

Beyond the resolution of tissue typing serology, HLA class I antigens display a certain level of structural microheterogeneity, that allows their subdivision into subtypes. The structure of these subtypes shows that multiple mechanisms operate in the generation of HLA polymorphism and suggests possible evolutionary pathways for subtype diversification. In addition, subtype polymorphism critically affects cellular allorecognition and antigen presentation to self-restricted T cells. These properties are used to define the structure and diversity of T-cell epitopes. In this review, José López de Castro discusses the nature and evolution of this polymorphism and its modulation of antigen recognition by cytolytic T lymphocytes.

Structure, function, and disease association of HLA-B27.

The specificity of antigen presentation by HLA-B27 and its modulation by subtype polymorphism can be understood from knowledge of the three-dimensional structure of this molecule and from biochemical analysis of bound peptides. These studies reveal the basis for the sharing of T cell determinants that might be critical to linkage of multiple subtypes to spondyloarthropathies. The mechanism of this association remains unknown, but involvement of T lymphocytes and HLA-B27 on antigen-presenting cells appears likely. Thus, a direct link between peptide presentation by HLA-B27 and its role in disease is emerging.

Structural polymorphism and function of HLA-B27.

Over the past year, new subtypes have been described and significant advances have been made in understanding peptide binding to HLA-B27. These developments help in interpreting how antigen presentation is modulated by HLA-B27 polymorphism, in determining the similarities and differences in T cell antigenicity among subtypes, and in finding the basis of HLA-B27 evolution. New insights into the pathogenetic link between enteric bacteria and HLA-B27 come from demonstration of a direct relationship between gut flora and joint inflammation in transgenic rats and from involvement of HLA-B27 in modulating bacterial invasion of mammalian cells. The association between HLA-B27 and spondyloarthropathy may be related to antigen-presenting specificity, but a different mechanism, presentation of a B27-derived peptide by class II antigens, may contribute to the pathogenesis of acute anterior uveitis, emphasizing the need for a comprehensive understanding of the structure, antigenicity, and function of HLA-B27.