Breaking the rules: the unconventional recognition of HLA-B27 by CD4+ T lymphocytes as an insight into the pathogenesis of the spondyloarthropathies

L. H. Boyle and J. S. Hill Gaston

University of Cambridge School of Clinical Medicine, Department of Medicine, Cambridge, UK


    Abstract
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 Abstract
 Association of HLA-B27 with...
 Structure and function of...
 Unique features of HLA-B27
 Animal models of...
 Recognition of unusual forms...
 A role for HLA-B27-specific...
 A model of the...
 Future directions
 References
 
Despite extensive research, it remains unclear why a small proportion of HLA- B27+ individuals develop spondyloarthropathies (SpA). Because the function of HLA-B27, as a major histocompatibility complex (MHC) class I molecule, is peptide presentation to CD8+ T cells, research has concentrated on the role of HLA-B27 as a restriction element for CD8+ cytotoxic T lymphocytes in pathogenesis. However, findings in the B27-transgenic animal models, together with the identification of unusual processing and presentation features of HLA-B27, have raised alternative hypotheses for the pathogenic role of HLA-B27. One such hypothesis is that HLA-B27 can be recognized by CD4+ T lymphocytes. Here we report the identification of such unusual cells, which break the conventional rules of MHC restriction, and propose a model for the role of such CD4+ T cells in SpA.

KEY WORDS: Major histocompatibility complex, Spondyloarthropathy, HLA-B27, T lymphocytes.


    Association of HLA-B27 with the spondyloarthropathies
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 Abstract
 Association of HLA-B27 with...
 Structure and function of...
 Unique features of HLA-B27
 Animal models of...
 Recognition of unusual forms...
 A role for HLA-B27-specific...
 A model of the...
 Future directions
 References
 
The strongest known association between a human leucocyte antigen (HLA) allele and a disease is that of HLA-B27 and the inflammatory spondyloarthropathies (SpA). The first linkage between SpA and this major histocompatibility complex (MHC) class I molecule was reported in 1973, and it was originally observed that 90% of patients with ankylosing spondylitis (AS) were HLA-B27+ compared with approximately 8% of healthy controls [1, 2]. Several SpA have now been connected to the HLA-B27 gene with various degrees of relative risk, including reactive arthritis, psoriatic spondylitis, spondylitis of inflammatory bowel disease, and juvenile spondylitis. Insight into the potential importance of an environmental factor triggering SpA has come from studies of reactive arthritis, where initiation of disease is linked to infection with various bacteria, including Chlamydia, Yersinia, Salmonella and Shigella [3, 4]. However, despite extensive research it remains unclear why a small proportion of HLA-B27+ individuals develop SpA and what the role of HLA-B27 is in its pathogenesis.


    Structure and function of HLA-B27
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 Abstract
 Association of HLA-B27 with...
 Structure and function of...
 Unique features of HLA-B27
 Animal models of...
 Recognition of unusual forms...
 A role for HLA-B27-specific...
 A model of the...
 Future directions
 References
 
The primary function of HLA-B27 as an MHC molecule is the presentation of peptides to T lymphocytes to induce protective immune responses. Conventionally, MHC class I heavy chains, such as HLA-B27, form a heterodimer with ß2-microglobulin (ß2m) and present endogenous peptides to CD8+ T lymphocytes, whereas CD4+ T lymphocytes recognize MHC class II molecules presenting peptides. As the function of HLA-B27 is peptide presentation to CD8+ T cells, much work has concentrated on identifying HLA-B27-restricted CD8+ T cells from SpA patients in order to understand why HLA-B27 elicits aberrant immune responses in individuals with SpA. This work has identified CD8+ T cells that show (i) autoreactive responses to HLA-B27 [5], (ii) HLA-B27-restricted responses to intracellular bacteria [57] and (iii) HLA-B27-restricted responses to self peptides, which can cross-react with HLA-B27-restricted responses to peptides derived from an infectious agent [8].

However, as some of these responses can be observed in both HLA-B27+ patients and HLA-B27+ healthy individuals, their relevance to disease remains obscure. Therefore, the focus has moved from the role of HLA-B27 as a restriction element for CD8+ T cells and has turned to other features of HLA-B27 which set it apart from other MHC class I molecules.


    Unique features of HLA-B27
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 Association of HLA-B27 with...
 Structure and function of...
 Unique features of HLA-B27
 Animal models of...
 Recognition of unusual forms...
 A role for HLA-B27-specific...
 A model of the...
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HLA-B27 heterodimers are both structurally and functionally similar to other human MHC class I molecules in their ability to present peptides to CD8+ T cells. However, there are a number of unusual features of HLA-B27 that make it unique amongst HLA molecules (Fig. 1Go). HLA-B27 heavy chains are unlike other HLA molecules, and similar to mouse MHC class I molecules, in their continued association with calnexin after complexing with ß2m [9]. In the absence of essential components of the MHC processing pathway, most MHC class I heavy chains are retained in the endoplasmic reticulum (ER) and degraded. However, HLA-B27 can be expressed on the surface of cells in the absence of two essential processing molecules, the transporter associated with antigen processing (TAP) complex [1012], and tapasin [13], both of which are involved in the loading of peptides onto MHC class I molecules. This independence of HLA-B27 is further highlighted by its ability to be expressed in the presence of the adenoviral ER retention protein E3/19K [14], which binds to TAP and acts as a tapasin inhibitor, preventing MHC class I/TAP association [15]. The independence of HLA-B27 from TAP and tapasin is possibly due to empty HLA-B27 heterodimers remaining stable long enough to gain peptide from alternative sources in the ER. Another unique characteristic of HLA-B27, attributed to its B pocket, is its slow folding in the ER, which leads to the accumulation of misfolded forms of the HLA-B27 heavy chain, even in the presence of an intact antigen-processing pathway [16]. This misfolding results in the increased degradation of HLA-B27 heavy chains in the ERGo.



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FIG. 1. Unique features of HLA-B27. Like all MHC class I molecules, HLA-B27 heterodimers can be expressed on the surface of cells, and bind antigenic peptide (1). However, there are a number of unique features in the processing of HLA-B27. HLA-B27 heavy chains remain complexed with calnexin after ß2m binding (2). HLA-B27 heavy chains undergo increased misfolding compared with other MHC class I alleles (3). HLA-B27 can be expressed in the absence of tapasin and TAP molecules (4). Unlike other MHC class I molecules, empty HLA-B27 heterodimers (5) and free HLA-B27 heavy chains (6) can be stably expressed on the surface of cells. These free HLA-B27 heavy chains can form an unusual homodimer structure (7), dependent on the free cysteine residue at position 67 in the peptide-binding groove.

 


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FIG. 2. Possible forms of HLA-B27 which could be implicated in the pathogenesis of the SpA. HLA-B27 forms a conventional HLA-B27 heterodimer structure associated with ß2m with peptide present in the groove (1). However, the HLA-B27 heterodimer can also be stably expressed as an empty HLA-B27 heterodimer, devoid of peptide in the groove (2). Empty HLA-B27 heterodimers can further dissociate into free HLA-B27 heavy chains (3). These free HLA-B27 heavy chains may be stabilized by presenting a peptide (4) or by forming free HLA-B27 heavy chain homodimers (5), dependent on the free cysteine residue at position 67. This free HLA-B27 heavy chain homodimer could also be able to present peptide (6).

 
Another unusual structural feature of HLA-B27 is the presence of an unpaired cysteine residue at position 67 (C67) in the B pocket of the peptide binding groove. Although this residue is found in other HLA molecules that are not associated with SpA, the presence of C67 together with other residues in the HLA-B27 binding pocket, such as the unique lysine at position 70, may result in a unique structural property of HLA-B27 [17, 18]. In addition to a conventional HLA-B27 heterodimer structure presenting a nonamer peptide [19], it has been demonstrated that a number of unusual forms of HLA-B27 can be expressed on the surface of cells in vitro , including empty HLA-B27 heterodimers devoid of peptide [20], and HLA-B27 molecules containing peptides up to 33 amino acids long [21, 22]. In the absence of ß2m, free HLA-B27 heavy chains can form a disulphide-bonded homodimeric structure, dependent on C67 [23]. HLA-B27 homodimers react with two MHC class I-specific monoclonal antibodies—HC10, which recognizes free MHC class I heavy chains [24], and W6/32, which recognizes correctly folded MHC class I heavy chains usually associated with ß2m [25, 26]—but homodimers are not currently thought to be recognized by the HLA-B27 heterodimer-specific monoclonal antibody ME1 [23]. This unusual form of HLA-B27 can also be expressed on the surface of cells [23, 27].

C67 not only plays an essential role in the formation of HLA-B27 homodimers but also appears to be essential in the thermostability of the HLA-B27 heterotrimer complex. Mutation of C67 to serine (C67S) alters some of the peptides bound to the HLA-B27 molecule and can destabilize B*2705 peptide complexes [28, 29].

Despite the identification of these various unique properties of HLA-B27, it remains unclear which, if any, of these properties of HLA-B27 are involved in the pathogenesis of SpA.


    Animal models of spondyloarthropathies
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 Abstract
 Association of HLA-B27 with...
 Structure and function of...
 Unique features of HLA-B27
 Animal models of...
 Recognition of unusual forms...
 A role for HLA-B27-specific...
 A model of the...
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 References
 
HLA-B27 transgenic animal models of SpA have been extremely important in deducing various factors likely to be involved in HLA-B27 linked disease. These animal models have implicated the expression of unconventional forms of HLA-B27 in the development of arthritis in two ways. Firstly, in HLA-B27-transgenic mice, the development of arthritis appears to be associated with the expression of free HLA-B27 heavy chains not associated with ß2m. This correlation was based on the observation that free HLA-B27 heavy chains are expressed in HLA-B27 transgenic mice lacking mouse ß2m (B27+2m-/-) and in HLA-B27+2m-/- mice made transgenic for human ß2m (B27+2m-/-2m+). Both these transgenic strains develop disease, whereas disease is absent from HLA-B27-transgenic mice that express mouse ß2m normally (B27+2m+) [30]. The reduction of arthritis in transgenic mice by in vivo treatment with HC10 supported the idea that free HLA-B27 heavy chain expression is associated with development of disease in these animals [30]. Secondly, in HLA-B27-transgenic rats, disease susceptibility is related to a high copy number of the HLA-B27 gene [31]. This gives rise to very high levels of expression of HLA-B27 and, together with the slow kinetics of HLA-B27 folding, may present opportunities for abnormal forms of HLA-B27 to be generated, and give rise to arthritis.

Evidence from both HLA-B27+-transgenic mice and rats supports the importance of an infectious agent triggering inflammatory arthritis, because animals maintained in germ-free environments did not develop arthritis but the disease phenotype appeared on subsequent transfer to conventional colonies [32, 33]. In addition, evidence from these animal models suggests that TAP molecules are not essential for SpA development as TAP2 polymorphisms in the HLA-B27+ transgenic rats do not alter the development of disease [34]. (Although this paper has been retracted, the data concerning the effect of the polymorphic rat TAP2 on inflammatory disease in the HLA-B27 transgenic rats remain valid [35].) In addition, inflammatory disease in the B27+2m-/- 2m+ mice occurs regardless of whether the TAP1 gene is present [36]. Nevertheless, MHC-bound peptides appear to have a role in the development of disease, as replacement of approximately 90% of endogenous peptides bound to HLA-B27 with an influenza-derived HLA-B27 binding peptide, expressed as a transgene, reduced the prevalence of arthritis in disease-prone HLA-B27+ rats [37].

Although the function of HLA-B27 is peptide presentation to CD8+ T lymphocytes, studies in HLA-B27+-transgenic animals have also raised the possibility that the role of HLA-B27 in joint disease may not simply be as a restriction element for CD8+ T cells. CD8+ T cells do not appear to be pivotal in disease induction, as B27+2m-/- mice with extremely low levels of CD8+ T cells still develop inflammatory disease [33] and adoptive transfer studies from disease-prone HLA-B27+ transgenic rats to HLA-B27+ nude rats showed that CD4+ T cells are more efficient than CD8+ T cells in transferring inflammatory disease [38]. In some circumstances MHC class II molecules, which are conventionally recognized by CD4+ T cells, are not required for disease development, as MHC class II-negative, HLA-B27+-transgenic mice still develop spontaneous disease [39]. Therefore, it appears to be unlikely that the presentation of a HLA-B27-derived peptide by MHC class II molecules to CD4+ T cells is important in the initiation of disease. One hypothesis that would explain all these findings is that CD4+ T cells can recognize HLA-B27, and are involved in disease initiation.


    Recognition of unusual forms of HLA-B27 by CD4+ T lymphocytes
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 Abstract
 Association of HLA-B27 with...
 Structure and function of...
 Unique features of HLA-B27
 Animal models of...
 Recognition of unusual forms...
 A role for HLA-B27-specific...
 A model of the...
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On the basis of evidence from the HLA-B27-transgenic animal models and the identification of an HLA-B27 homodimer structure which might mimic the structure of a MHC class II molecule [40, 41], it has been hypothesized that CD4+ T cells, which conventionally recognize MHC class II molecules, can recognize the MHC class I molecule HLA-B27, breaking the conventional rules of MHC restriction. Although there is no evidence from the animal models that HLA-B27 itself is directly recognized by CD4+ T cells, the recognition of HLA-B27 by CD4+ T cells would explain the apparent requirement for both HLA-B27 and CD4+ T cells in the HLA-B27-transgenic animal models of SpA. It would also be consistent with other observations in humans, such as the large numbers of CD4+ T cells in diseased joints [42, 43]. Therefore, we attempted to isolate CD4+ T cells with the ability to recognize HLA-B27 from HLA-B27+ AS patients and HLA-B27+ healthy individuals. Using a co-culture system, we isolated a number of CD4+ T cells from HLA-B27+ AS patients with the unconventional ability to recognize unusual forms of HLA-B27 expressed on several cell lines with defects in their ability to process antigens: T2 [lacks TAP and low molecular weight proteins (LMP) involved in generating antigenic peptides], 721.220 (tapasin-defective) and C1R (hemizygous for MHC).

Recognition of empty HLA-B27 heterodimers
One type of HLA-B27-reactive CD4+ T cell isolated only responded to HLA-B27 expressed on the processing-defective cell lines and did not respond to HLA-B27 expressed on peripheral blood mononuclear cells (PBMC) and Epstein–Barr virus (EBV)-transformed cell lines [44]. Therefore it was unlikely that a conventional form of HLA-B27 was recognized. However, inhibition of responses with W6/32 and ME1 suggested that a heterodimeric structure of HLA-B27 was recognized. This pattern of responses could be explained if the HLA-B27 heterodimers recognized on the antigen processing defect cell lines contained a peptide that was absent from HLA-B27+ intact cell lines or were devoid of peptide. Further studies implied that ‘empty’ or ‘peptide-receptive’ HLA-B27 heterodimers were recognized by these CD4+ T cells, as increased responses were observed to T2-B27 incubated at 26°C (which stabilizes the expression of heterodimers devoid of peptide), and the responses to 721.220-B27 and C1R-B27 were inhibited by high-affinity B27-binding peptides derived from HIV and influenza virus. Therefore, one type of HLA-B27-reactive CD4+ T cells appears to recognize an aberrant form of HLA-B27, namely empty HLA-B27 heterodimers.

Recognition of an abnormally folded HLA-B27 complex
A second type of HLA-B27-reactive CD4+ T cell appeared not to be dependent on defects in antigen processing because increased responses were observed to T2-B27 reconstituted with TAP molecules. Interestingly, the responses to HLA-B27 were inhibited by W6/32 but not ME1, suggesting the recognition of an unconventional form of HLA-B27, i.e. not a heterodimeric structure of HLA-B27. The CD4+ T cells also responded to HLA-B27+ EBV-transformed cell lines and HLA-B27+ PBMC. These results suggest the recognition of an epitope expressed on an abnormally folded form of HLA-B27, possibly a homodimeric structure of HLA-B27. Whether this structure also presents an antigenic peptide to the CD4+ T cells is unknown: the fact that responses are considerably enhanced by the transfection of TAP suggests that there is a requirement for a TAP-transported antigenic peptide. Increased expression of W6/32-reactive HLA-B27 homodimers has been reported in T2-B27 transfected with TAP molecules [27], possibly because TAP-dependent peptides stabilize this form of HLA-B27.

In addition to HLA-B27-reactive CD4+ T cells, we have also isolated CD4+ T cells that can respond to HLA-A2 as an alloantigen, suggesting that the ability of CD4+ T cells to interact with a MHC class I antigen is not confined to HLA-B27. This is in agreement with previous studies that have isolated HLA-A2-restricted CD4+ T cells from humans [4550]. However, the isolation of HLA-B27-reactive CD4+ T cells from HLA-B27+ SpA patients has not been documented previously.


    A role for HLA-B27-specific CD4+ T cells in spondyloarthropathies?
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 Abstract
 Association of HLA-B27 with...
 Structure and function of...
 Unique features of HLA-B27
 Animal models of...
 Recognition of unusual forms...
 A role for HLA-B27-specific...
 A model of the...
 Future directions
 References
 
The identification of HLA-B27-reactive CD4+ T cells in HLA-B27+ AS patients raises the possibility that unconventional recognition of HLA-B27 is important in the pathogenesis of SpA. Because these HLA-B27-specific CD4+ T cells break the conventional rules of MHC restriction, it is possible they are involved in the pathogenesis by producing an autoimmune response to HLA-B27. In support of the notion that MHC class I-restricted CD4+ T cells might be able to cause disease, MHC class I-restricted CD4+ T cells, derived from MHC class II-deficient mice, induce an aggressive inflammatory bowel disease in congenic, immunodeficient mice [51]; gut inflammation is also a prominent feature of some HLA-B27-associated SpA and in HLA-B27-transgenic rats.

To isolate these HLA-B27-reactive CD4+ T cells, we used a panel of stimulator cells with defects in antigen presentation. However, it is possible that in vivo normal antigen-presenting cells acquire similar properties to the mutant cells used to isolate HLA-B27-specific CD4+ T cells. At present, there are three scenarios in which MHC class I antigen processing pathway defects occur in humans.

Due to genetic defects in essential components of the MHC class I processing pathway. A variety of such abnormalities are found in malignant transformations of cells, in which reduced expression of HLA class I molecules is observed. Mutations in the ß2m gene [5254] and defects in the expression of TAP and LMP genes [55], many of which are due to abnormal transcriptional regulation, are frequently observed in a variety of tumours.

During infection with intracellular pathogens. Alterations to the MHC class I processing pathway are most commonly observed during viral infection. For example, the TAP complex is targeted by viruses in order to decrease MHC class I expression on the cell surface. The human cytomegalovirus US6 protein [5658] and the herpes simplex virus ICP47 protein [59] both target the TAP complex to inhibit peptide transport. This inevitably results in expression of altered MHC class I molecules, including empty molecules.

As a result of the action of cytokines. The ability of both a viral interleukin (IL)-10 homologue and human IL-10 to efficiently down-regulate the expression of the LMP2 and TAP1 genes, which results in inefficient transport of peptides into the ER [60], indicates that constitutive [61] or induced [62] IL-10 production or altered cytokine patterns could lead to deficiencies in the MHC class I processing pathway.

Currently, only limited information is available concerning any associations between SpA and components of the antigen-processing pathway, such as the LMP [63, 64] and TAP molecules [6568]. In addition, although there is no evidence that viral infection is associated with initiation of the SpA [6972], in view of the association between the SpA and bacterial infections [7375] and the importance of the normal intestinal flora in the induction of arthritis in HLA-B27-transgenic rodents [32, 33], it is worth considering the possibility that intracellular bacteria can subvert MHC expression in similar ways to viruses. Indeed, the intracellular bacterium Chlamydia trachomatis, one of the bacteria associated with reactive arthritis, has been shown to alter MHC expression [76]. In addition, in the light of the identification of non-MHC genetic susceptibility factors on chromosomes 1p, 2q, 6p, 9q, 10q, 16q and 19q that are associated with the SpA [77], it is worth considering that genetic or environmental alterations in cytokine production could result in abnormal MHC class I processing. If these or other similar antigen-processing deficiencies were present in HLA-B27+ individuals, even temporarily in a single tissue, this could lead to the expression of altered forms of HLA-B27 in vivo which would be available for recognition by HLA-B27-specific CD4+ T cells.

The weaker association of B*2706 and B*2709 with AS might also be accounted for by subtle effects of these polymorphisms [78, 79]. Although B*2709 does not have a significant effect on the ratio of native to ß2m-free HLA-B27 heavy chains when compared with B*2705 [78], it is possible that the subtle effects observed on peptide binding to these alleles may result in altered stability of B*2709 or B*2706 heterodimers, especially in the presence of defects in antigen presentation (i.e. B*2706 and B*2709 may not be expressed stably as empty B27 heterodimers in the presence of antigen-processing defects).


    A model of the role of HLA-B27-specific CD4+ T cells in spondyloarthropathies
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 Abstract
 Association of HLA-B27 with...
 Structure and function of...
 Unique features of HLA-B27
 Animal models of...
 Recognition of unusual forms...
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 A model of the...
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Although it cannot be determined at this stage whether HLA-B27-specific CD4+ T cells play any role in the pathogenesis of the SpA, a model of how unconventional recognition of HLA-B27 by CD4+ T cells could trigger inappropriate immune responses can be proposed (Fig. 3Go). Defects in antigen processing, such as those described above, would result in the expression of altered forms of HLA-B27 in vivo, which would be available for recognition by immune system cells, including the HLA-B27-specific CD4+ T cells that we have identified. Initial activation of these cells would probably require traffic of antigen-presenting cells bearing altered forms of HLA-B27 (e.g. infected dendritic cells) to regional lymph nodes, but subsequent recruitment of primed cells to the joint or to entheses [80] would result in their activation if altered forms of HLA-B27 were present locally. It is interesting to speculate that the mechanical and physiological properties of the enthesis (mechanical stretch, hypoxia) might in some way predispose to the expression of altered forms of HLA-B27, and this idea needs to be explored experimentally. Activation of these CD4+ T cells would result in the induction of an inflammatory response, with recruitment of inflammatory cells, such as macrophages and other phagocytic cells, to entheses or synovium, where they could be activated and release inflammatory cytokines. The release of IL-8, tumour necrosis factor {alpha} and IL-12 at this site would activate the vascular endothelium, increase vascular permeability and activate further lymphocytes and natural killer (NK) cells. Alternatively, if these CD4+ T cells provide help for B cells, this could result in the high levels of IgA seen in SpA, particularly if the T cells also secrete transforming growth factor ß. Subsequent complement activation would result in the lysis of cells by the membrane attack complex, cytokine and prostaglandin release and the attraction of inflammatory cells, and likewise tissue injury from products of activated leucocytes. The persistence of antigen, in this case unusual forms of HLA-B27, would result in continued inflammation leading to tissue destruction.



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FIG. 3. Model of the role of HLA-B27-specific CD4+ T cells in the SpA. Alterations to the MHC class I processing pathway, for example by viral or bacterial infection, genetic mutations or altered cytokine patterns (1), could lead to altered expression of HLA-B27 in vivo. Unusual forms, including empty HLA-B27 heterodimers and abnormally folded HLA-B27 complexes such as homodimers, could be recognized by HLA-B27-specific CD4+ T cells (2). This would lead to proliferation of CD4+ T cells and release of cytokines (3), which would attract inflammatory cells such as macrophages and NK cells into the disease site (4). Release of cytokines by inflammatory cells would result in tissue damage and further recruitment of inflammatory cells (5).

 
It is worth considering how HLA-B27-specific CD4+ T cells might be selected in the thymus. It is possible that unusual forms of HLA-B27 are present in the thymus [81] and that CD4+ T cells can be positively selected on such forms and are therefore HLA-B27-restricted CD4+ T cells. This assumes that the interaction between CD4+ T cells and HLA-B27 in the thymus is not so strong as to result in deletion. Indeed, expression of unusual forms of MHC class I molecules, due to antigen-processing defects, can result in positive selection of CD8+ T cells in mice [82]. In humans lacking MHC class II expression, a small population of CD4+ T cells develop, suggesting that CD4+ T cells can be selected in the absence of MHC class II molecules [83]. Alternatively, the HLA-B27-reactive CD4+ T cells could actually be MHC class II-restricted CD4+ T cells selected on MHC class II molecules in the thymus, but with the ability to cross-react in the periphery with unusual forms of MHC class I molecules that appear due to external influences. In these circumstances, self-tolerance would be broken because the CD4+ T cells would not have the opportunity to encounter altered forms of HLA-B27 in the thymus. In support of such cross-reactivity, unusual alloreactive CD8+ T cells have been described which are HLA-B27-specific but can also cross-react with HLA-DR2 molecules [84, 85].

HLA-B27-specific CD4+ T cells may be present in both healthy and diseased HLA-B27+ individuals, but may have been identified in the HLA-B27+ patients because they were present at a higher frequency. A deficiency in peripheral tolerance [86], such as a shortage in regulatory T cells, lack of inhibition induced by cytotoxic T lymphocyte antigen 4 (CTLA-4), or inappropriate co-stimulation due to an environment factor may also allow these autoreactive CD4+ T cells to proliferate, increase in number and result in an inappropriate inflammatory response. Indeed, deficiencies in mechanisms of peripheral tolerance can trigger autoimmune diseases. For example, a deficiency in CD4+CD25+ regulatory T cells can trigger intestinal inflammation [87] and autoimmune thyroiditis [88].


    Future directions
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To understand fully the critical role of HLA-B27 in the pathogenesis of the SpA, much research is required. Our findings further highlight the unusual properties of HLA-B27, and we propose that altered recognition of HLA-B27 and the induction of inappropriate immune responses may play a role in this disease. Many questions remain to be answered following our identification of HLA-B27-specific CD4+ T cells. The first step in determining whether HLA-B27-specific CD4+ T cells are involved in disease is to measure whether these cells are present at higher frequencies in HLA-B27+ SpA patients compared with HLA-B27+ healthy individuals. Similarly, if these cells are involved in disease, increased numbers should be present at affected sites. Therefore, comparing the frequency of HLA-B27-specific CD4+ T cells in the blood and synovial fluid would allow conclusions about the likely relevance of these CD4+ T cells in disease. Because HLA-B27 transgenic animal models of SpA describe the involvement of both HLA-B27 and CD4+ T cells, it may also be worthwhile to determine whether HLA-B27-specific CD4+ T cells can be isolated from these rodents.

Although we have highlighted three possible mechanisms (genetic defects, infection and cytokines) in which alterations to the MHC class I processing pathway can occur, it will also be important to determine which factors can result in unusual expression of B27 in vivo. As noted above, the influence that the environment of the enthesis might have on HLA-B27 processing and expression is worth further experimentation.

Whilst we have described cells that are activated by unusual forms of HLA-B27, apparently through their T-cell receptors, other receptors might also interact with these forms. Indeed, Allen et al. [89] have recently identified interactions between HLA-B27 and members of the killer immunoglobulin receptor (KIR) and the immunoglobulin-like transcripts (ILT). These receptors are expressed by NK cells and monocytes, as well as by T cells, and widen the possible ways in which abnormal forms of HLA-B27 might be involved in SpA pathogenesis (Fig. 4Go).



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FIG. 4. Possible effect of the recognition of HLA-B27 by NK receptors on the CD4+ T cell response to HLA-B27. The recognition of a form of HLA-B27, such as a homodimer, by KIRs or ILTs expressed on NK cells could result in an altered pattern of cytokine release from the NK cells, which in turn could affect the response produced by the HLA-B27-reactive CD4+ T cells. Alternatively, the HLA-B27-reactive CD4+ T cells may themselves express KIR or ILTs, and binding of HLA-B27 by the KIR or ILTs could also influence the response produced by the T cells.

 
In conclusion, the identification of CD4+ T cells in HLA-B27+ AS patients that recognize unusual forms of HLA-B27, such as empty HLA-B27 heterodimers and homodimeric structures of HLA-B27, offers a novel pathogenic mechanism for SpA as an alternative to the assumption that an HLA-B27-associated disease will be initiated by classical MHC class I-restricted CD8+ T cells. CD4+ T cells, which break the conventional rules of MHC restriction, could be responsible for the immune responses that initiate and maintain chronic inflammatory disease in joints and entheses.


    Acknowledgments
 
We thank the Arthritis Research Campaign and GlaxoSmithKline for funding this work.


    Notes
 
Correspondence to: L. H. Boyle, Department of Medicine, Box 157, Level 5, Addenbrooke's Hospital, Cambridge CB2 2QQ, UK. E-mail: lhb22{at}medschl.cam.ac.uk Back


    References
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 Abstract
 Association of HLA-B27 with...
 Structure and function of...
 Unique features of HLA-B27
 Animal models of...
 Recognition of unusual forms...
 A role for HLA-B27-specific...
 A model of the...
 Future directions
 References
 

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Submitted 8 May 2002; Accepted 8 August 2002