Expression and function of HLA-B27 in lipid-linked form: Implications for cytotoxic T lymphocyte-induced apoptosis signal transduction

Major histocompatibility complex (MHC) class I antigen-restricted cytotoxic T lymphocytes (CTL) kill their target cells not only by inducing irreversible membrane damage but also by triggering a programmed suicide cascade (apoptosis) in target cells. Recent evidence suggests that MHC class I antigens are involved in apoptosis signal transduction in T cells. Therefore, it is possible that MHC class I antigens are also responsible for CTL-induced signal transduction in target cells leading to apoptosis. To test this hypothesis, we have expressed HLA-B27 in Chinese hamster ovary (CHO) cells in a phosphatidyl inositol (PI) anchored form. The expressed Pl-anchored HLA-B27 (PI-B27), a 42-kDa molecule which can be cleaved off the cell surface by Pi-specific phospholipase C, can function as an MHC restriction and antigen presentation element for specific CTL. Furthermore, PI-B27 transfectant CHO cells undergo rapid DNA fragmentation when pulsed with the appropriate peptide and treated with specific CTL, suggesting that the cytoplasmic and transmembrane domains of the heavy chain of class I MHC molecules are not required in CTL-induced apoptosis signal transduction in target cells.     

Co-expression of the human HLA-B27 class I antigen and the E3/19K protein of adenovirus-2 in insect cells using a baculovirus vector

We have expressed the human MHC class I HLA-B27 antigen, human beta 2-microglobulin, and the E3/19K protein of adenovirus-2 in Spodoptera frugiperda insect cells (Sf9) by using the Autographa californica nuclear polyhedrosis virus. All genes were inserted under the strong polyhedrin promoter in the vector pVL941. The proteins were expressed at high levels, ranging from 1 to 8 mg protein per 3 x 10(9) cells. Both a full-length and a truncated form of HLA-B27 were expressed. The latter was terminated at the border of the membrane-spanning segment at the extracellular side of the membrane. The HLA-B27 antigens and the E3/19K protein showed considerable heterogeneity with respect to glycosylation. Only a small fraction of HLA-B27 was assembled (less than 5%) with beta 2-microglobulin. Nevertheless, we were able to find the heavy chain at the cell surface, and by co-infection with the recombinant virus for beta 2-microglobulin we observed an increase in cell surface expression. The E3/19K protein of adenovirus-2 blocks cell surface expression of HLA class I antigens in human cells and has a similar effect in insect cells. In contrast to beta 2-microglobulin it assembles efficiently to the heavy HLA-B27 chain, both the full-length and truncated forms. The E3/19K protein does not stimulate assembly of HLA-B27-beta 2-microglobulin. Due to these problems of assembly and the heterogeneity of glycosylation we predict that it will be difficult to use HLA antigens produced in insect cells for X-ray crystallographic studies.     

Luminal bacterial antigen-specific CD4+ T-cell responses in HLA-B27 transgenic rats with chronic colitis are mediated by both major histocompatibility class II and HLA-B27 molecules

Rats transgenic (TG) for the human major histocompatibility complex (MHC) class I HLA-B27 and beta2-microglobulin genes develop chronic colitis under specific pathogen-free (SPF) but not sterile (germ-free, GF) conditions. We investigated the role of antigen-presenting molecules involved in generating immune responses by CD4+ mesenteric lymph node (MLN) cells from colitic HLA-B27 TG rats to commensal enteric micro-organisms. All TG MLN cells expressed HLA-B27. A higher level of MHC class II was expressed on cells from TG rats, both SPF and GF, compared to non-TG littermates. In contrast, rat MHC class I expression was lower on TG than non-TG cells. Both TG and non-TG antigen presenting cells (APC) pulsed with caecal bacterial antigens induced a marked interferon-gamma (IFN-gamma) response in TG CD4+ T lymphocytes but failed to stimulate non-TG cells. Blocking MHC class II on both TG and non-TG APC dramatically inhibited their ability to induce TG CD4+ T cells to produce IFN-gamma. Blocking HLA-B27 on TG APC similarly inhibited IFN-gamma responses. When the antibodies against MHC class II and HLA-B27 were combined, no APC-dependent IFN-gamma response was detected. These data implicate both native rat MHC class II and TG HLA-B27 in CD4+ MLN T-cell IFN-gamma responses to commensal enteric microflora in this colitis model.     

The biochemistry and immunology of non-canonical forms of HLA-B27

HLA-B27 (B27) is strongly associated with the spondyloarthritides. B27 is expressed at the cell surface of antigen presenting cells (APC) both as canonical β2m-associated and non-canonical β2m-free heavy chain (FHC) forms which include B27 dimers (termed B27₂). B27 FHC forms arise in an endosomal compartment from recycling β2m-associated B27. Formation of cell surface FHC dimers is critically dependent on an unpaired reactive cysteine 67 in the α1 helix of the class I heavy chain. HLA-B27 also form redox-inducible β2m-associated dimers on exosomes and apoptosing cells. By contrast with cell surface expressed cysteine 67-dependent heavy chain dimers these dimers are dependent on a cytoplasmic cysteine 325 for their formation.     

Strong association of HLA-B27 heavy chain with beta 2-microglobulin

Monoclonal antibody TG1 recognizes specifically antigens HLA-B27, B7, B22 and B17 on cell surface in cytotoxicity and cytofluorometry tests. When cell detergent extracts were subjected to SDS PAGE under mild conditions (no heating and no reduction of the sample) followed by Western blotting, TG1 detected exclusively a complex of B27 heavy chains with β₂-microglobulin (as a 50 kDa band) whereas the other B-locus antigens (B7, B22, B17) were detected as free 43 kDa heavy chains under the same conditions. When the samples were boiled prior to SDS PAGE, TG1 detected again the 43 kDa free heavy chains of B7, B22 and B17 but no zone corresponding to B27 could be detected indicating that the epitope in free B27 chains is more sensitive to denaturation by SDS. Thus, our main finding is that the interaction of HLA-B27 heavy chain with β₂-microglobulin appears to be stronger than that of the other HLA-B chains. The resistance of the HLA-B27/β₂-microglobulin complex to the SDS dissociation is strikingly similar to the behavior of MHC class II molecules under similar conditions. Thus, it may be speculated that HLA-B27 complexes can be also more stable than other MHC class I molecules under more physiological dissociative conditions (e.g. in endosomal compartments). This feature might potentially influence antigen presentation by HLA-B27 and contribute to the well known disease linkage of HLA-B27.     

Frequency of HLA-B27 subtypes in a Danish population and in Danish patients with ankylosing spondylitis

Polymerase chain reaction in combination with sequence-specific oligonucleotide probes were used to analyze nine HLA-B27 subtypes among 51 healthy I HLA-B27 positive Danish blood donors and 30 Danish HLA-B27 positive patients with ankylosing spondylitis (AS). In the group of healthy Danes we found two subtypes, B*2705 (90.2%) and B*2702 (9.8%), however, among the AS patients only the B*2705 subtype was detected. We did not find a significant evidence for associations between AS and a particular HLA-B27 subtype in a Danish population.     

An extra molecule in addition to human tapasin is required for surface expression of beta2m linked HLA-B4402 on murine cell

Murine MHC class I can be readily expressed on the surface of human cell lines, but human class I molecules are expressed on mouse cells at a reduced level. Both human beta-2-microglobulin (beta(2)m) and tapasin (Tpn) have been demonstrated to be required for proper human MHC class I surface expression. Here we report that besides beta(2)m and tapasin, an extra unidentified component is also critical for the expression of certain human class I alleles. By covalently linking HLA-B4402 heavy chain to beta(2)m (beta(2)m-B44) a pre-assembled class I molecule has been created, which can be efficiently expressed and travel to the surface in human cells. In spite of being able to express inside cells, the linked beta(2)m-B44 molecule does not express on the surface of a murine fibroblast. Further investigation shows that lack of appearance on the surface is not due to quick degradation of unloaded class I, since provision of HLA-B4402 binding peptide could not rescue impaired surface expression. Co-expression with human tapasin does not rescue the defect excluding tapasin as the critical component for expression and indicating that a novel component of human origin is required for efficient surface expression of beta(2)m-B44 in murine cells. Surprisingly, not only did the beta(2)m-B44 construct fail to express on murine cells but also the surface expression of native murine MHC class I Kb was greatly reduced in transfected cells. It is likely that the expressed linked chain competitively associates with a component of class I processing in murine cells, reducing the exit rate of assembled mouse class I molecules. The results together suggest an unknown mechanism, which leads to the trapping of class I molecules in the ER.     

Interactions of HLA-B27 with the peptide loading complex as revealed by heavy chain mutations

MHC class I heavy chains assemble in the endoplasmic reticulum with beta(2)-microglobulin and peptide to form heterotrimers. Although full assembly is required for stable class I molecules to be expressed on the cell surface, class I alleles can differ significantly in their rates of, and dependencies on, full assembly. Furthermore, these differences can account for class I allele-specific disparities in antigen presentation to T cells. Recent studies suggest that class I assembly is assisted by an elaborate complex of proteins in the endoplasmic reticulum, collectively referred to as the peptide loading complex. In this report we take a mutagenesis approach to define how HLA-B27 molecules interact with the peptide loading complex. Our results define subtle differences between how B27 mutants interact with tapasin (TPN) and calreticulin (CRT) in comparison to similar mutations in other mouse and human class I molecules. Furthermore, these disparate interactions seen among class I molecules allow us to propose a spatial model by which all class I molecules interact with TPN and CRT, two molecular chaperones implicated in facilitating the binding of high-affinity peptide ligands.     

Lymphoblastoid cells express HLA-B27 homodimers both intracellularly and at the cell surface following endosomal recycling

The MHC class I allele HLA-B27 is very strongly associated with development of autoimmune spondyloarthritis, although the disease mechanism remains unknown. Class I molecules classically associate in the endoplasmic reticulum (ER) with beta2-microglobulin (beta(2)m) and antigenic peptides for cell surface expression and presentation to T cells. We have previously shown that HLA-B27 is capable of forming beta(2)m-free disulfide-bonded homodimers in vitro. Here we show that HLA-B27 forms disulfide-bonded homodimers in vivo by two distinct pathways. HLA-B27 homodimers form in the ER but appear unable to egress to the cell surface in human cells. Cell surface HLA-B27 homodimers are abundantly expressed in a variety of lymphoid cell lines. Experiments with inhibitors indicate that HLA-B27 homodimers can arise from cell-surface heterodimers via an endosome-dependent recycling pathway. HLA-B27 homodimer expression on the cell surface of 721.220 is dependent on the unpaired cysteine(67) and is inhibited by restoration of tapasin function or by incubation with peptides that bind strongly to HLA-B27 heterodimers. Cell surface expressed HLA-B27 homodimers are likely to be immunologically reactive ligands for NK family immunoreceptors and, hence, could play a pathogenic role in spondyloarthritis.     

HLA-B*27 subtypes in Northern and Northeastern Thais, Karens, and Bamars determined by a high-resolution PCR-SSP technique

Human leukocyte antigens (HLA), class I, are a group of antigens expressed on most nucleated cell surfaces. They transport endogenous peptides to the cell surface for recognition by T-cell receptors. Their functions are involved in immune responses. Many diseases are associated with HLA alleles, especially HLA-B*27 that is strongly associated with ankylosing spondylitis (AS). HLA-B*27 consists of 42 subtypes. Different subtypes of HLA-B*27 were reported in different ethnic groups of AS patients. In this study, a high-resolution polymerase chain reaction–sequence-specific primer technique has been developed to define all the HLA-B*27 subtypes with a total of 29 primer mixtures. Two of the primer mixes were used to detect the HLA-B*27 -specific group, and 27 primer mixes were used to identify 42 subtypes ( B*2701–B*2721 and B*2723–B*27 43). The HLA-B*27 -group-specific primers have been tested in unrelated healthy subjects; 846 Northeastern Thais (NET), 334 Northern Thais (NT), 264 Karens, and 310 Bamars. Sixty-three NET (phenotype frequency, PF = 7.4%), 24 NT (PF = 7.1%), 5 Karens (PF = 1.8%), and 12 Bamars (PF = 3.9%) were positive for HLA-B*27 . Only B*2704 was found in Karens, whereas B*2704 , B*2705/37/39 , B*2706 , and B*2707 were found in NET and NT. In Bamars, B*2704 , B*2705/37/39 , B*2706 , and B*2725 were found. The distribution of HLA-B*27 subtypes was compared with other studies in Asian and Caucasian populations. Significant differences of the distribution of HLA-B*27 subtypes were found in most of the populations. This study established a simple technology for HLA-B*27 subtyping and provided basic information for anthropology and further studies in disease associations.     

Guilt by association: HLA-B27 and ankylosing spondylitis

The remarkable association between HLA-B27 and ankylosing spondylitis (AS) remains an enigma. While previous reviews have discussed the controversies surrounding the involvement of bacteria in the etiology of this disease and the sequence variability between subtypes of HLA-B27, concepts of disease mechanism remain ill-defined. In this article Richard Benjamin and Peter Parham synthesize new data on the structure and function of HLA class I molecules into possible mechanisms that might underly the pathogenesis of AS.     

Strong association of HLA-B27 heavy chain with β2-microglobulin

Monoclonal antibody TG1 recognizes specifically antigens HLA-B27, B7, B22 and B17 on cell surface in cytotoxicity and cytofluorometry tests. When cell detergent extracts were subjected to SDS PAGE under mild conditions (no heating and no reduction of the sample) followed by Western blotting, TG1 detected exclusively a complex of B27 heavy chains with β₂-microglobulin (as a 50 kDa band) whereas the other B-locus antigens (B7, B22, B17) were detected as free 43 kDa heavy chains under the same conditions. When the samples were boiled prior to SDS PAGE, TG1 detected again the 43 kDa free heavy chains of B7, B22 and B17 but no zone corresponding to B27 could be detected indicating that the epitope in free B27 chains is more sensitive to denaturation by SDS. Thus, our main finding is that the interaction of HLA-B27 heavy chain with β₂-microglobulin appears to be stronger than that of the other HLA-B chains. The resistance of the HLA-B27/β₂-microglobulin complex to the SDS dissociation is strikingly similar to the behavior of MHC class II molecules under similar conditions. Thus, it may be speculated that HLA-B27 complexes can be also more stable than other MHC class I molecules under more physiological dissociative conditions (e.g. in endosomal compartments). This feature might potentially influence antigen presentation by HLA-B27 and contribute to the well known disease linkage of HLA-B27.     

Structurally diverse forms of HLA-B27 molecules are displayed in vivo in a cell type-dependent manner

The formation of a trimeric complex, composed of heavy chain (HC), beta(2)-microglobulin (beta(2)m) and antigenic peptide, is generally believed to be a prerequisite for the expression of HLA class I molecules at the cell surface in vivo. Therefore, a possible role in immunological processes for HC/beta(2)m complexes devoid of peptide has not been seriously considered. Using a novel HLA-B*2705-transgenic rat model and monoclonal antibodies that distinguish between structurally different forms of HLA-B27 molecules, we demonstrate here that class I molecules which appear to lack antigenic peptides are expressed in abundance on a variety of cell types in lymphoid organs. These results imply a role for structurally diverse, possibly empty, MHC molecules in physiological T cell selection which has so far not been sufficiently appreciated.     

HLA-B27 misfolding and ankylosing spondylitis

Understanding how HLA-B27 contributes to the pathogenesis of spondyloarthritis continues to be an important goal. Current efforts are aimed largely on three areas of investigation; peptide presentation to CD8T cells, abnormal forms of the HLA-B27 heavy chain and their recognition by leukocyte immunoglobulin-like receptors on immune effector cells, and HLA-B27 heavy chain misfolding and intrinsic biological effects on affected cells. In this chapter we review our current understanding of the causes and consequences of HLA-B27 misfolding, which can be defined biochemically as a propensity to oligomerize and form complexes in the endoplasmic reticulum (ER) with the chaperone BiP (HSPA5/GRP78). HLA-B27 misfolding is linked to an unusual combination of polymorphisms that identify this allele, and cause the heavy chain to fold and load peptides inefficiently. Misfolding can result in ER-associated degradation (ERAD) of heavy chains, which is mediated in part by the E3 ubiquitin ligase HRD1 (SYVN1), and the ubiquitin conjugating enzyme UBE2JL. Upregulation of HLA-B27 and accumulation of misfolded heavy chains can activate ER stress signaling pathways that orchestrate the unfolded protein response. In transgenic rats where HLA-B27 is overexpressed, UPR activation is prominent. However, it is specific for heavy chain misfolding, since overexpression of HLA-B7, an allele that does not misfold, fails to generate ER stress. UPR activation has been linked to cytokine dysregulation, promoting lL-23, IFNβ, and lL-1α production, and may activate the IL-23/IL-17 axis in these rats. IL-1α and IFNβ are pro- and anti-osteoclastogenic cytokines, respectively, that modulate osteoclast development in HLA-B27-expressing transgenic rat monocytes. Translational studies of patient derived cells expressing HLA-B27 at physiologic levels have provided evidence that ER stress and UPR activation can occur in peripheral blood, but this has not been reported to date in isolated macrophages. Inflamed gastrointestinal tissue reveals evidence for HLA-B27 misfolding, ERAD, and autophagy, without acute UPR activation. A more complete picture of conditions that impact HLA-B27 folding and misfolding, the full spectrum and time course of consequences of ER stress, and critical cell types involved is needed to understand the role of HLA-B27 misfolding in spondyloarthritis pathogenesis.     

MHC class I dimer formation by alteration of the cellular redox environment and induction of apoptosis

Many MHC class I molecules contain unpaired cysteine residues in their cytoplasmic tail domains, the function of which remains relatively uncharacterized. Recently, it has been shown that in the small secretory vesicles known as exosomes, fully folded MHC class I dimers can form through a disulphide bond between the cytoplasmic tail domain cysteines, induced by the low levels of glutathione in these extracellular vesicles. Here we address whether similar MHC class I dimers form in whole cells by alteration of the redox environment. Treatment of the HLA-B27-expressing Epstein-Barr virus-transformed B-cell line Jesthom, and the leukaemic T-cell line CEM transfected with HLA-B27 with the strong oxidant diamide, and the apoptosis-inducing and glutathione-depleting agents hydrogen peroxide and thimerosal, induced MHC class I dimers. Furthermore, induction of apoptosis by cross-linking FasR/CD95 on CEM cells with monoclonal antibody CH-11 also induced MHC class I dimers. As with exosomal MHC class I dimers, the formation of these structures on cells is controlled by the cysteine at position 325 in the cytoplasmic tail domain of HLA-B27. Therefore, the redox environment of cells intimately controls induction of MHC class I dimers, the formation of which may provide novel structures for recognition by the immune system.     

Threonine 80 on HLA-B27 confers protection against lysis by a group of natural killer clones

Recognition of major histocompatibility complex (MHC) class I molecules on target cells by natural killer (NK) cells inhibits NK cell-mediated lysis. Although it is known that this inhibitory effect is regulated by MHC polymorphism, the precise structural determinants remain undefined. Based on the capacity of different HLA-C and HLA-B motifs specifically to inhibit cytotoxicity of some NK clones, three different NK cell specificities (NK1, NK2 and NK3) have been described. In this study, the recognition of HLA-B27 by NK clones has been analyzed using C1R cells transfected with different HLA-B27 subtypes as target cells. Cytotoxicity was inhibited by the HLA-B*2705, -B*2701 -B*2703, -B*2704 and -B*2706 alleles, but not by -B*2702. This subtype is distinguished from the other B27 subtypes by the presence of isoleucine instead of threonine at position 80. Direct involvement of this residue was assessed by showing that site-directed mutagenesis of Thr80 to Ile80 in HLA-B*2705 reverted the NK protective effect of HLA-B*2705. Based on these data, we suggest that Thr80 could act as a single residue conferring target cell protection from lysis by a group of NK clones, tentatively designated NK4.     

Rapid acidification and alkylation: redox analysis of the MHC class I pathway

The technique of rapid acidification and alkylation can be used to characterise the redox status of oxidoreductases, and to determine numbers of free cysteine residues within substrate proteins. We have previously used this method to analyse interacting components of the MHC class I pathway, namely ERp57 and tapasin. Here, we have applied rapid acidification/alkylation as a novel approach to analysing the redox status of MHC class I molecules. This analysis of the redox status of the MHC class I molecules HLA-A2 and HLA-B27, which is strongly associated with a group of inflammatory arthritic disorders referred to as Spondyloarthropathies, revealed structural and conformational information. We propose that this assay provides a useful tool in the study of in vivo MHC class I structure.     

HLA-B27等位基因和亚型与强直性脊柱炎关系研究进展

大量的研究证明,强直性脊柱炎(AS)是与人类白细胞抗原(HLA)相关性最强的疾病。AS的发病与HLA-B27阳性密切相关,并与B7、B13、B40等几个等位基因有一定关系。HLA-B位点有42个等位基因,其中HLA-B27具有高度多态性,含有22个以上的亚型,不同亚型的碱基序列间只有个别差异。B27亚型在AS患者中的分布因地区和种族上的差别而不同,在中国主要以B2704和B2705为主,但以B2705分布最广。这几年大量的人B27转基因鼠实验证明AS与B27的关联性。     

Influence of infection of cells with bacteria associated with reactive arthritis on the peptide repertoire presented by HLA-B27

Reactive arthritis (ReA) after infections with various gram-negative bacteria is strongly associated with the MHC class I molecule HLA-B27. It is supposed that the B27 molecule itself plays a role in the pathogenesis of ReA by presenting antigenic peptides to cytotoxic T lymphocytes. The peptide repertoires presented by Salmonella-, Shigella- and non-infected cells were compared to identify such peptides. From the peptides isolated from the B27 molecules of these cells, profiles were generated by reversed-phase chromatography and peaks present in the profiles from infected cells but not in profiles from non-infected cells were studied for their peptide compositions. Some sequences with identity to those in human histone H3, human ribosomal protein S17 and the heavy chain of HLA-B27 itself were detected only in profiles from infected cells. All peptides identified from infected cells contained the B*2705 peptide-binding motif. The data suggest that HLA-B27-positive cells infected with ReA-inducing bacteria show an increased presentation of certain self-peptides. There was no evidence for altered peptide-binding specificity of B27 after infection. However, the interpretations were hampered by the variation in peptide presentation between different experiments.     

HLA-B27 and the pathogenesis of spondyloarthropathies

The association of HLA-B27 with ankylosing spondylitis, a chronic inflammatory disease of the axial skeleton, and other spondyloarthropathies, is among the strongest of an MHC antigen and any disease. Yet, the basis for this association remains unknown. In this review the main current hypotheses concerning the pathogenetic role of HLA-B27 will be discussed. They focus on three molecular properties of the molecule: (1) its peptide-presenting specificity, (2) its slow folding and tendency to misfold, and (3) its capacity to form covalent heavy chain homodimers amenable to recognition by leukocyte receptors. On the basis of the peptide specificity spondyloarthropathies would be triggered through T-cell autoimmunity against a self-ligand of HLA-B27 elicited by a cross-reactive foreign antigen. HLA-B27 misfolding would trigger disease through activation of inflammatory pathways following induction of endoplasmic reticulum stress, thus independently of antigen presentation. Recognition of heavy chain homodimers by leukocyte receptors might be involved in disease through immunomodulation of both innate and adaptive responses to arthritogenic pathogens. None of these hypotheses can yet satisfactorily account for the pathogenesis of spondyloarthritides. It is proposed that the pathogenetic role of HLA-B27 will eventually be explained through a global understanding of its biology, in which the various features of this molecule are envisaged as inter-dependent in their contribution to disease.