Avidity dictates the lytic capacity of human cytolytic T lymphocyte clones with similar fine specificity against murine cells expressing HLA-B27 antigen

The role of the avidity of human CTL in the recognition and lysis of murine P815 cells expressing HLA-B27.1 Ag has been examined. Seven B27-specific alloreactive CTL clones were tested for their ability to lyse a B27.1+-P815 transfectant clone 1-7E, obtained after cotransfection of P815-HTR cells with HLA-B27.1 and human beta 2-microglobulin genes. The expression level of HLA-B27.1 on 1-7E cells was comparable to that on a human lymphoblastoid cell line, as determined by flow cytometry. Of the seven CTL clones used, CTL 1, 26, and 29 displayed the same fine specificity as established with a panel of target cells expressing six structurally different HLA-B27 variants. However, CTL 1 and 29 were of higher avidity than CTL 26, in that the lysis of human target cells by only this latter clone was inhibited by an anti-CD8 mAb. Based on the same criteria, CTL 2, 15, and 48 possessed the same or very similar fine specificity, but CTL 48 was of higher avidity than CTL 2 or 15. The seventh clone, CTL 40, was of a different fine specificity and its lysis of human target cells was also inhibited by the same anti-CD8 mAb. Only those clones whose lysis of human targets could not be inhibited by anti-CD8 antibody were able to lyse the 1-7E murine transfectants. These results indicate that, for human CTL clones with identical or very similar fine specificity, only those of higher avidity are able to lyse P815 murine cells expressing the HLA-B27 antigen. The lysis of HLA-B27.1+-murine transfectants by relevant clones was inhibited by anti-CD8 antibody. This result strongly suggests that the relative contribution of CD8 in stabilizing the interaction between human CTL and HLA-B27+-murine target cells is more significant than with human target cells.     

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.     

Alloreactive cytotoxic T-lymphocyte-defined HLA-B7 subtypes differ in peptide antigen presentation

We investigated T-cell-defined HLA-B7 subtypes using cDNA sequencing, analysis of bound peptides, and reactivity with a panel of alloreactive cytotoxic T-lymphocyte (CTL) clones. Three subtypes (HLA-B*0702, HLA-B*0703, and HLA-B*0705) differ in nucleotide and predicted amino acid sequence. CTL reactivity and pooled peptide sequencing show that these three HLA-B7 subtypes bind distinct but overlapping sets of peptides. In particular B*0702 expresses D pocket residue Asp 114 and binds peptides with P3 Arg, whereas B*0705 expresses D pocket residue Asn 114 and binds peptides with P3 Ala, Leu, and Met. Consistent with different peptide-binding specificities, three alloreactive CTL differentiate between cells expressing B*0702, B*0703, and B*0705 by detecting specific peptide/HLA-B7 complexes. In contrast, three other T-cell-defined HLA-B7 subtypes are identical to HLA-B*0702. The B*0702-expressing cell lines are differentiated by two of ten CTL clones. One CTL clone differentiates B*0702-expressing cells by their ability to present peptide antigen. Thus differences in peptide presentation can explain differential CTL recognition of cell lines expressing structurally identical and variant HLA-B7.     

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

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

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.     

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.     

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

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

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.     

One allogeneic cytolytic T lymphocyte clone distinguishes three different HLA-B27 subtypes: identification of amino acid residues influencing the specificity and avidity of recognition

The HLA-B27 antigen may be divided into at least three subgroups, designated HLA-B27.1, -B27.2, and -B27.3, by specific cytolytic T lymphocytes. In an attempt to explore the functional relevance of HLA polymorphism, an alloimmune cytolytic T cell clone T3+, T8+, T4- has been characterized, which displays a distinct reactivity pattern with each one of the three HLA-B27 subtypes. This cell kills both B27.1- and B27.2- but not B27.3-positive targets. Its lytic efficiency is greater with B27.1 than with B27.2 cells. The clone does not recognize either B7-positive targets or most B27-negative cells. But HLA-B40-bearing cells are lysed, albeit with significantly less efficiency than any B27-positive targets. The differences in killing ability for B27.1, B27.2, and B40 are also evident in cold-target inhibition studies, indicating that a) B27.1 cells can efficiently inhibit lysis of B27.2 and B40 targets, b) B27.2 cells inhibit the lysis of B40 but not of B27.1 targets, and c) B40 cells do not inhibit B27.1 or B27.2 target lysis. In addition, anti-T3 and anti-T8 antibodies are much more effective in inhibiting the lysis of B27.2 targets than that of B27.1-positive cells, suggesting that the observed differences in killing efficiency of the various targets are due to the fact that the tightness of the effector-target interaction is affected by the structural changes between the different HLA antigens. A correlation of the reactivity pattern of this T cell clone with the known amino acid sequences of the HLA-B27, HLA-B40, and HLA-B7 antigens suggests that the clone recognizes a conformational determinant contributed to by residues within the segments 149-156 and 67-83. Those in the former segment appear to be an essential portion of this determinant, whereas polymorphism in the region 67-83 has a modulating effect on the reactivity of the effector but does not abrogate recognition.     

Structural analysis of an HLA-B27 population variant, B27f. Multiple patterns of amino acid changes within a single polypeptide segment generate polymorphism in HLA-B27

The structure of a new HLA-B27 variant, B27f, distinguishable from other HLA-B27 subtypes by isoelectric focusing and serologic criteria, has been established by comparative peptide mapping and radiochemical sequence analysis. HLA-B27f differs from the major B27.1 subtype in three clustered amino acid replacements: Asp74, Asp77, and Leu81 in B27.1 are changed to Tyr74, Asn77, and Ala81, respectively in B27f. This pattern of differences is analogous to that of HLA-B27.2 in that this subtype also differs from B27.1 in multiple clustered substitutions within the same segment. Thus, polymorphism within the HLA-B27 system is being achieved by introducing different sets of amino acid changes within a particular short segment of the alpha 1 domain. The most likely mechanism for the introduction of multiple changes within this segment is a nonreciprocal recombination event, such as gene conversion. The structural analogies and ethnic distribution of B27f and B27.2 as compared with those of B27.3, and B27.4 support a dynamic model of HLA-B27 evolution in which polymorphism has been created after the separation of the major ethnic groups. In this model, a Caucasian branch would be characterized by subtypes differing from B27.1 in a few changes within the alpha 1 domain, which were probably generated by single genetic steps. An Oriental branch would include those subtypes which differ from B27.1 by changes in both alpha 1 and alpha 2, involving multiple genetic steps for their generation.     

Role of binding pockets for amino-terminal peptide residues in HLA-B27 allorecognition.

The peptide binding site of HLA-B27 and other class I Ag consists of a series of pockets that bind peptide side chains. Two of these pockets interact with the amino-terminal peptide residue (pocket A) and with the highly conserved second residue (pocket B). In this study, the role of pockets A and B in HLA-B27-specific T cell allorecognition has been analyzed. Four HLA-B27 mutants with single or double changes in pocket B (24T----A, 45E----M, 67C----V, and 24,67T,C----A,V) and three mutants with single changes in pocket A (163E----T, 167W----S, and 171Y----H) were constructed by site-directed mutagenesis and expressed in HMy2.C1R cells after DNA-mediated gene transfer. These transfectants were used as target cells in cytotoxicity assays with a series of HLA-B27-specific CTL. All the mutations analyzed affected allorecognition by a significant proportion of the CTL tested, but no single change abrogated recognition by all CTL. The global effects of each mutation on allorecognition were comparable to one another, except for the effect of the change at position 67, which was smaller. The behavior of individual CTL with the mutants was very diverse, ranging from CTL that did not recognize most of the mutants to CTL recognizing all of them. Thus, some alloreactive CTL can withstand drastic alterations in pockets A and B. Two CTL showed heteroclytic effects towards the V67 and M45 mutants. CTL behavior with the H171 mutant was closely parallel to that with the B*2703 subtype, having a single Y----H change at position 59. This parallelism correlates with the similar role of Tyr59 and Tyr171 in establishing hydrogen bonds with the amino termini of HLA-B27-bound peptides. The results demonstrate that altering the structure of pockets that interact with the amino-terminal first and second residues of HLA-B27-bound peptides significantly affects recognition by alloreactive CTL, and they strongly suggest widespread peptide involvement in HLA-B27 allorecognition.     

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).     

Multiple epitopes on human and murine cells expressing HLA-B7 as defined by specific murine cytotoxic T cell clones

Eleven cytotoxic T lymphocyte (CTL) clones were derived from C57BL/6 spleen cells immunized with HLA-B7 expressing human lymphoblastoid cell lines. Reactivity against HLA-B7 was initially established because the clones lysed 2 target cells that shared only HLA-B7 with the immunizing cell line and they did not lyse five other cell lines that were HLA-B7 negative but expressed other class I or class II antigens found on the immunizing cell. Six of the clones were subsequently shown to lyse all tested HLA-B7-positive B and T lymphoid cell lines, peripheral blood lymphocytes, and a murine L cell that expressed HLA-B7 as a consequence of DNA-mediated gene transfer. On the basis of the inability of the clones to lyse a panel of HLA-B7-negative cell lines, up to 18 other class I antigens could be eliminated as being cross-reactively recognized. However, two of the clones recognized a single HLA-B7-negative cell line. It is suggested that in these cases the clones were cross-reactively recognizing the HLA-B27 or HLA-B40 antigens that were present on these target cells. The remaining five CTL clones failed to lyse one out of seven tested HLA-B7-positive lymphoid lines (either RPMI-1788 or DR1B) and failed to lyse peripheral blood lymphocytes from one out of three tested HLA-B7-positive individuals. These five clones also did not recognize the HLA-B7-positive murine L cell. However, based on analysis with a large target cell panel, the reactivity pattern of these five clones could only be correlated with recognition of HLA-B7. This conclusion is further supported by antibody-blocking studies to be reported elsewhere. As before, lysis of single HLA-B7-negative target cells by two of the clones could be ascribed to recognition of HLA-B27 or HLA-B40. The results show that murine clones raised against HLA-B7 exhibit a high degree of specificity for determinants that are unique or largely confined to the HLA-B7 alloantigen. In addition, these clones define different antigenic determinants on the molecule. Thus, such clones appear to be excellent candidates for use as human tissue typing reagent. The results further show that there is a strong correlation between recognition of particular HLA-B7-positive human cell lines and recognition of the HLA-B7 expressing murine L cell. Possible reasons for such a correlation and their relationship to the general phenomenon of CTL recognition are discussed.     

Differential recognition by human cytotoxic T cell clones of human M1 fibroblasts transfected with an HLA-B7 gene (JY150) suggests the existence of two different HLA-B7 alleles in the cell line JY (HLA-A2,2;B7,7;Cw-,-;DR4,w6)

We have used a panel of human HLA-B7-specific CTL clones to identify an HLA-B7 gene (JY150) transfected into human M1 fibroblasts (M1/B7). Only a subset of the CTL clones recognized the M1/B7 cells, whereas all CTL clones recognized the donor of the B7 gene, the cell line JY (HLA-A2,2;B7,7;Cw-,-;DR4,w6). Analysis of the fine specificity of these CTL clones was performed by testing the reactivity on M1 cells transfected with an HLA-B27K gene and on a panel of cell lines typed for HLA-B7 subtypes (variants). These results, combined with one-dimensional IEF analysis of the M1/B7 cells and the B7 subtypes, indicated that the differential recognition by the CTL clones of the transfected gene was not caused by aberrant expression of the gene itself or due to the absence of critical accessory molecules on the M1 fibroblast cells. Our data suggest that the widely used HLA-B7 reference cell line JY is not homozygous at the HLA-B locus, but contains two different B7 alleles encoding the B7.2 and B7.4 subtypes.     

A novel HLA-B27 allele maps B27 allospecificity to the region around position 70 in the alpha 1 domain.

There are six known HLA-B alleles that share the HLA-B27 allospecificity, yet differ by one to six amino acid substitutions. Each of these B27 alleles can be readily assigned by one of the six representative IEF patterns. Two unrelated individuals, LH and HS, express B27 Ag that appear to be identical by IEF, but an HLA-B27 alloreactive CTL clone I-73 was found to react differently with these cells, suggesting these B27 molecules are not identical. We sequenced polymerase chain reaction-amplified B27 cDNA clones obtained from HS and compared its deduced amino acid sequence (B27-HS) with the B27 sequence of LH (B27-LH) which was previously designated the B*2701 allele. B27-HS and B27-LH differ by eight amino acids; three in alpha 1 domain and five in alpha 2 domain. These amino acid substitutions of B27-HS altered T cell recognition but not the B27 serologic epitope or IEF pattern. B27-HS differs from the six known B27 alleles by five to eight amino acid substitutions, and thus it represents the seventh allele of the HLA-B27 Ag family. This novel B27 allele might have been derived from a gene conversion event. Previously, two amino acid residues at positions 70 and 97 were suggested to be specific for B27 Ag family. B27-HS now reveals that Lys at position 70 is specific for B27 but Asn at position 97 is not. We propose that the region around position 70 might be crucial in determining the B27 serologic epitope and possibly in peptide Ag binding. This study also demonstrates that class I molecules of the same Ag specificity sharing an indistinguishable IEF pattern are not necessarily identical, and indicates that only the definitive determination of primary structure would identify all the class I alleles that are functionally relevant in regard to alloreactivity, T cell restriction, and disease association.     

Recognition of HLA-B27 by mouse cytotoxic T-cell clones: a transgenic mouse

As a basis for the characterization of mouse T cells involved in the recognition of xenogeneic HLA molecules, a panel of HLA-B27-reactive cytotoxic T-cell clones was generated upon stimulation by cells from HLA-B27-transgenic mice. The HLA-B27-induced T-cell response was found to comprise two categories of clones: some recognizing HLA-B27 independent of H-2 molecules expressed by the target cells (unrestricted clones), others recognizing HLA-B27 in an H-2 restricted manner. The unrestricted clones exhibited diverse specificities, as judged from their various cross-reactivities with other xenogeneic (HLA) or allogeneic (H-2) molecules. In addition, although most of the unrestricted clones were able to react with both mouse and human HLA-B27-transgenic mice. The HLA-B27 induced T-cell which reacted only with HLA-B27-positive mouse, and not human cells. These findings illustrate that both H-2-restricted and unrestricted T cells with diverse species contribute to HLA-B27-xenorecognition.     

Animal models of HLA-B27-associated diseases

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

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.     

Asymmetric selection of T cell antigen receptor alpha- and beta-chains in HLA-B27 alloreactivity.

Endogenous peptides constitutively bind to class I MHC Ag and are thought to be integral parts of allospecific T cell epitopes. However, allospecific TCR can recognize structural features of the alloantigen as foreign. To define some crucial parameters determining HLA-B27 allorecognition, the structure of TCR alpha- and beta-chains from HLA-B27-specific CTL was analyzed. A strategy, based on V alpha and V beta family-specific oligonucleotides, was used for specific amplification and direct sequencing of TCR-alpha and -beta cDNA. We observed nonrandom usage of V beta segments and recurrent structural motifs within beta-chain junctional regions. In contrast, no structural restrictions were apparent among alpha-chains, even from CTL clones of related fine specificity. These results indicate an asymmetric contribution of TCR alpha- and beta-chains to HLA-B27 allospecificity among the CTL clones analyzed. They suggest recognition of multiple peptides and involvement of beta-chain junctional regions in recognizing shared motifs among some of these peptides.     

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.