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P Bowness - One of the best experts on this subject based on the ideXlab platform.
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th17 cells expressing kir3dl2 and responsive to hla b27 homodimers are increased in ankylosing spondylitis
Journal of Immunology, 2011Co-Authors: P Bowness, Andrew J Mcmichael, J Shaw, A Ridley, Isabel Wongbaeza, A T Chan, Myles Fleming, Fraser Cummings, S KollnbergerAbstract:CD4 Th cells producing the proinflammatory cytokine IL-17 (Th17) have been implicated in a number of inflammatory arthritides including the spondyloarthritides. Th17 development is promoted by IL-23. Ankylosing spondylitis, the most common spondyloarthritis (SpA), is genetically associated with both HLA-B27 (B27) and IL-23R polymorphisms; however, the link remains unexplained. We have previously shown that B27 can form H chain dimers (termed B272), which, unlike classical HLA-B27, bind the killer-cell Ig-like receptor KIR3DL2. In this article, we show that B272-expressing APCs stimulate the survival, proliferation, and IL-17 production of KIR3DL2+ CD4 T cells. KIR3DL2+ CD4 T cells are expanded and enriched for IL-17 production in the blood and synovial fluid of patients with SpA. Despite KIR3DL2+ cells comprising a mean of just 15% of CD4 T in the peripheral blood of SpA patients, this subset accounted for 70% of the observed increase in Th17 numbers in SpA patients compared with control subjects. TCR-stimulated peripheral blood KIR3DL2+ CD4 T cell lines from SpA patients secreted 4-fold more IL-17 than KIR3DL2+ lines from controls or KIR3DL2− CD4 T cells. Strikingly, KIR3DL2+ CD4 T cells account for the majority of peripheral blood CD4 T cell IL-23R expression and produce more IL-17 in the presence of IL-23. Our findings link HLA-B27 with IL-17 production and suggest new therapeutic strategies in ankylosing spondylitis/SpA.
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interaction of hla b27 homodimers with kir3dl1 and kir3dl2 unlike hla b27 heterotrimers is independent of the sequence of bound peptide
European Journal of Immunology, 2007Co-Authors: S Kollnberger, Antoni Chan, Meiyi Sun, L Chen, Cynthia Wright, Kati Di Gleria, Andrew J Mcmichael, P BownessAbstract:HLA-B27 can form beta-2 microglobulin (β2m)-associated heterotrimers (HLA-B27) and β2m-free homodimers (B272). Here, we study the role of complexed peptide in the interaction of these forms of B27 with the killer cell immunoglobulin (Ig)-like receptors KIR3DL1 and KIR3DL2 and with Ig-like transcripts LILRB1 and LILRB2. HLA-B27 tetramers complexed with three of five different naturally processed self peptides and three of seven pathogen-derived epitopes bound to KIR3DL1-expressing transfectants and NK cells. Heterotrimeric complexes containing peptides with charged amino acids at position 8 did not bind to KIR3DL1; however, studies with analogue peptides demonstrated that these are not the only peptide residues involved in binding. KIR3DL1 ligation by HLA-B27 inhibited NK cell IFN-γ production in a peptide-dependent fashion. B27 but not HLA-A2, B7 or B57 heavy chains formed homodimers in the presence of peptide epitopes. B272 bound to KIR3DL1, KIR3DL2 and LILRB2 but not LILRB1. KIR3DL2 ligation by B272 inhibited NK and T cell IFN-γ production. By contrast with HLA heterotrimers, B272 binding to KIR did not depend on the sequence of the bound peptide. Differences in KIR binding to classical HLA and B272 could be involved in the pathogenesis of spondyloarthritis.
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Use of HLA-B27 tetramers to identify low-frequency antigen-specific T cells in Chlamydia-triggered reactive arthritis
Arthritis Res Ther, 2004Co-Authors: Heiner Appel, S Kollnberger, P Bowness, Wolfgang Kuon, Maren Kuhne, Stefanie Kuhlmann, Andreas Thiel, Joachim SieperAbstract:Reports of the use of HLA-B27/peptide tetrameric complexes to study peptide-specific CD8^+ T cells in HLA-B27^+-related diseases are rare. To establish HLA-B27 tetramers we first compared the function of HLA-B27 tetramers with HLA-A2 tetramers by using viral epitopes. HLA-B27 and HLA-A2 tetramers loaded with immunodominant peptides from Epstein–Barr virus were generated with comparable yields and both molecules detected antigen-specific CD8^+ T cells. The application of HLA-B27 tetramers in HLA-B27-related diseases was performed with nine recently described Chlamydia -derived peptides in synovial fluid and peripheral blood, to examine the CD8^+ T cell response against Chlamydia trachomatis antigens in nine patients with Chlamydia -triggered reactive arthritis (Ct-ReA). Four of six HLA-B27^+ Ct-ReA patients had specific synovial T cell binding to at least one HLA-B27/ Chlamydia peptide tetramer. The HLA-B27/ Chlamydia peptide 195 tetramer bound to synovial T cells from three of six patients and HLA-B27/ Chlamydia peptide 133 tetramer to synovial T cells from two patients. However, the frequency of these cells was low (0.02–0.09%). Moreover, we demonstrate two methods to generate HLA-B27-restricted T cell lines. First, HLA-B27 tetramers and magnetic beads were used to sort antigen-specific CD8^+ T cells. Second, Chlamydia -infected dendritic cells were used to stimulate CD8^+ T cells ex vivo . Highly pure CD8 T cell lines could be generated ex vivo by magnetic sorting by using HLA-B27 tetramers loaded with an EBV peptide. The frequency of Chlamydia -specific, HLA-B27 tetramer-binding CD8^+ T cells could be increased by stimulating CD8^+ T cells ex vivo with Chlamydia -infected dendritic cells. We conclude that HLA-B27 tetramers are a useful tool for the detection and expansion of HLA-B27-restricted CD8^+ T cells. T cells specific for one or more of three Chlamydia -derived peptides were found at low frequency in synovial fluid from HLA-B27^+ patients with Ct-ReA. These cells can be expanded ex vivo , suggesting that they are immunologically functional.
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lymphoblastoid cells express hla b27 homodimers both intracellularly and at the cell surface following endosomal recycling
European Journal of Immunology, 2003Co-Authors: Lucy A Bird, S Kollnberger, Andrew J Mcmichael, Chen Au Peh, Tim Elliott, P BownessAbstract: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.
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cell surface expression and immune receptor recognition of hla b27 homodimers
Arthritis & Rheumatism, 2002Co-Authors: S Kollnberger, Meiyi Sun, Andrew J Mcmichael, Lucy A Bird, Christelle Retiere, Veronique M Braud, P BownessAbstract:Objective. HLA–B27 is capable of forming in vitro a heavy-chain homodimer structure lacking 2microglobulin. We undertook this study to ascertain if patients with spondylarthritis express 2-microglobulin– free HLA–B27 heavy chains in the form of homodimers and receptors for HLA–B27 homodimers. Methods. Expression of HLA–B27 heavy chains by mononuclear cells was analyzed by fluorescenceactivated cell sorter staining, Western blotting with the monoclonal antibody HC-10, and 2-dimensional isoelectric focusing. Fluorescence-labeled tetrameric complexes of HLA–B27 heavy-chain homodimers were constructed in which each dimer comprised one His-tagged heavy chain and one biotinylated heavy chain, and were used to stain patient and control mononuclear cells and transfected cell lines. Results. Patients with spondylarthritis expressed cell-surface HLA–B27 homodimers. Populations of synovial and peripheral blood monocytes, and B and T lymphocytes from patients with spondylarthritis, and controls carried receptors for HLA–B27 homodimers. Experiments with transfected cell lines demonstrated that KIR3DL1 and KIR3DL2, and immunoglobulin-like transcript 4 (ILT4), but not ILT2, are receptors for HLA–B27 homodimers. Conclusion. Patients with spondylarthritis express both HLA–B27 heavy-chain homodimers and receptors for HLA–B27 homodimers. This may be of significance with regard to disease pathogenesis.
Robert A. Colbert - One of the best experts on this subject based on the ideXlab platform.
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the human leukocyte antigen hla b27 peptidome in vivo in spondyloarthritis susceptible hla b27 transgenic rats and the effect of erap1 deletion
Molecular & Cellular Proteomics, 2017Co-Authors: Eilon Barnea, Tri M Tran, Nimman Satumtira, Martha L Dorris, Robert A. Colbert, Robert E Hammer, Dganit Melamed Kadosh, Yael Haimovich, Mylinh T Nguyen, Joel D. TaurogAbstract:HLA-B27 is a class I major histocompatibility (MHC-I) allele that confers susceptibility to the rheumatic disease ankylosing spondylitis (AS) by an unknown mechanism. ERAP1 is an aminopeptidase that trims peptides in the endoplasmic reticulum for binding to MHC-I molecules. ERAP1 shows genetic epistasis with HLA-B27 in conferring susceptibility to AS. Male HLA-B27 transgenic rats develop arthritis and serve as an animal model of AS, whereas female B27 transgenic rats remain healthy. We used large scale quantitative mass spectrometry to identify over 15,000 unique HLA-B27 peptide ligands, isolated after immunoaffinity purification of the B27 molecules from the spleens of HLA-B27 transgenic rats. Heterozygous deletion of Erap1, which reduced the Erap1 level to less than half, had no qualitative or quantitative effects on the B27 peptidome. Homozygous deletion of Erap1 affected approximately one-third of the B27 peptidome but left most of the B27 peptidome unchanged, suggesting the possibility that some of the HLA-B27 immunopeptidome is not processed in the presence of Erap1. Deletion of Erap1 was permissive for the AS-like phenotype, increased mean peptide length and increased the frequency of C-terminal hydrophobic residues and of N-terminal Ala, Ser, or Lys. The presence of Erap1 increased the frequency of C-terminal Lys and Arg, of Glu and Asp at intermediate residues, and of N-terminal Gly. Several peptides of potential interest in AS pathogenesis, previously identified in human cell lines, were isolated. However, rats susceptible to arthritis had B27 peptidomes similar to those of non-susceptible rats, and no peptides were found to be uniquely associated with arthritis. Whether specific B27-bound peptides are required for AS pathogenesis remains to be determined. Data are available via ProteomeXchange with identifier PXD005502.
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hla b27 alters the response to tumor necrosis factor α and promotes osteoclastogenesis in bone marrow monocytes from hla b27 transgenic rats
Arthritis & Rheumatism, 2013Co-Authors: Gerlinde Layhschmitt, Eva Y Yang, Grace Kwon, Robert A. ColbertAbstract:Objective To determine whether HLA–B27 expression alters the response of bone marrow monocytes from HLA–B27/human β2-microglobulin–transgenic (B27-Tg) rats to tumor necrosis factor α (TNFα) and, if so, whether this affects the cells involved in bone homeostasis. Methods Bone marrow monocytes were treated with RANKL or with TNFα to promote osteoclast formation. Osteoclasts were quantified by counting. Gene expression was measured using quantitative polymerase chain reaction analysis, and protein was detected by enzyme-linked immunosorbent assay, immunoblotting, or immunofluorescence. Effects of endogenously produced cytokines on osteoclast formation were determined with neutralizing antibodies. Results TNFα treatment enhanced osteoclast formation 2.5-fold in HLA–B27–expressing cells as compared to wild-type or to HLA–B7/human β2-microglobulin–expressing monocytes. TNFα induced ∼4-fold up-regulation of HLA–B27, which was associated with the accumulation of misfolded heavy chains, binding of the endoplasmic reticulum (ER) chaperone BiP, and activation of an ER stress response, which was not seen with HLA–B7. No differences were seen with RANKL-induced osteoclastogenesis. Enhanced interleukin-1α (IL-1α) production from ER-stressed bone marrow monocytes from B27-Tg rats was found to be necessary and sufficient for enhanced osteoclast formation. However, bone marrow monocytes from B27-Tg rats also produced more interferon-β (IFNβ), which attenuated the effect of IL-1α on osteoclast formation. Conclusion HLA–B27–induced ER stress alters the response of bone marrow monocytes from B27-Tg rats to TNFα, which is associated with enhanced production of IL-1α and IFNβ, cytokines that exhibit opposing effects on osteoclast formation. The altered response of cells expressing HLA–B27 to proinflammatory cytokines suggests that this class I major histocompatibility complex allele may contribute to the pathogenesis of spondyloarthritis and its unique phenotype through downstream effects involving alterations in bone homeostasis.
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enhanced phosphorylation of stat 1 is dependent on double stranded rna dependent protein kinase signaling in hla b27 expressing u937 monocytic cells
Arthritis & Rheumatism, 2012Co-Authors: Marja Ruuska, Kaisa Granfors, Robert A. Colbert, Anna S Sahlberg, Markus A PenttinenAbstract:Objective To study the phosphorylation of STAT-1 in HLA–B27–transfected human monocytic cells and the role of the signaling molecules double-stranded RNA–dependent protein kinase (PKR) and p38 in STAT-1 phosphorylation. Methods U937 human monocytic cell transfectants stably expressing wild-type HLA–B27 or mutated HLA–B27 heavy chains with amino acid substitutions in the B pocket were prepared. Mock-transfected cells were prepared using the antibiotic resistance vectors (pSV2neo or RSV5neo) alone. Phorbol myristate acetate–differentiated cells were stimulated with lipopolysaccharide (LPS) or infected with Salmonella enteritidis. The phosphorylation and expression levels of STAT-1 protein were detected by Western blotting and flow cytometry. Specific inhibitors were added in cell culture to study the role of PKR and p38 in STAT-1 phosphorylation. Results STAT-1 was constitutively highly phosphorylated on the tyrosine 701 residue in HLA–B27–positive monocytic cells when compared to control cells, even prior to stimulation with LPS or bacteria. This phenotype was associated with the expression of HLA–B27 heavy chains that misfold. In addition, phosphorylation of STAT-1 was dependent on PKR. Conclusion Our results show that STAT-1 tyrosine 701 is constitutively highly phosphorylated in the HLA–B27–expressing monocyte/macrophage cell line. Since phosphorylation of tyrosine 701 on STAT-1 is sufficient to induce interferon (IFN)–dependent genes, constitutive activity of this phosphorylation site may lead to the overexpression of IFN-dependent genes, as well as other STAT-1–dependent genes, in HLA–B27 monocyte/macrophages. Our results offer a mechanism by which B27 expression alone, without any external trigger, is potentially capable of inducing activation of STAT-1, a critical regulator of the inflammatory response.
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hla b27 up regulation causes accumulation of misfolded heavy chains and correlates with the magnitude of the unfolded protein response in transgenic rats implications for the pathogenesis of spondylarthritis like disease
Arthritis & Rheumatism, 2007Co-Authors: Matthew J Turner, Shuzhen Bai, Monica L Delay, Erin I Klenk, Robert A. ColbertAbstract:Objective HLA–B27 is implicated in the pathogenesis of spondylarthritis (SpA), yet the molecular mechanisms are incompletely defined. HLA–B27 misfolding has been associated with endoplasmic reticulum stress and activation of the unfolded protein response (UPR) in macrophages from HLA–B27/human β2-microglobulin–transgenic (B27-transgenic) rats. This study was performed to assess the mechanisms that drive activation of the HLA–B27–induced UPR and to determine whether splenocytes respond in a similar manner. Methods Splenocytes were isolated and bone marrow macrophages were derived from B27-transgenic and wild-type rats. Cells were treated for up to 24 hours with cytokines that induce class I major histocompatibility complex expression. HLA–B27 expression and misfolding were assessed by real-time reverse transcription–polymerase chain reaction, flow cytometry, and immunoblotting. Activation of the UPR was measured by quantifying UPR target gene expression and X-box binding protein 1 messenger RNA (mRNA) splicing. Results HLA–B27 mRNA up-regulation was accompanied by a dramatic increase in the accumulation of misfolded heavy chains and preceded robust activation of the UPR in macrophages. When macrophages were treated with various cytokines, the magnitude of the UPR correlated strongly with the degree of HLA–B27 up-regulation. In contrast, B27-transgenic splenocytes exhibited only low-level differences in the expression of UPR target genes after exposure to interferon-γ or concanavalin A, which resulted in minimal HLA–B27 up-regulation. Conclusion These results suggest that HLA–B27–associated activation of the UPR in macrophages is attributable to the accumulation of misfolded heavy chains, and that certain cell types may be more susceptible to the effects of HLA–B27 misfolding. Strategies that eliminate HLA–B27 up-regulation and/or the accumulation of misfolded heavy chains may be useful in evaluating the role of these events in the pathogenesis of SpA.
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hla b27 misfolding in transgenic rats is associated with activation of the unfolded protein response
Journal of Immunology, 2005Co-Authors: Matthew Turner, Joel D. Taurog, Robert A. Colbert, Dawn P Sowders, Monica L Delay, Rajashree Mohapatra, Shuzhen Bai, Judith A Smith, Jaclyn R BrandewieAbstract:The mechanism by which the MHC class I allele, HLA-B27, contributes to spondyloarthritis pathogenesis is unknown. In contrast to other alleles that have been examined, HLA-B27 has a tendency to form high m.w. disulfide-linked H chain complexes in the endoplasmic reticulum (ER), bind the ER chaperone BiP/Grp78, and undergo ER-associated degradation. These aberrant characteristics have provided biochemical evidence that HLA-B27 is prone to misfold. Recently, similar biochemical characteristics of HLA-B27 were reported in cells from HLA-B27/human β 2 -microglobulin transgenic (HLA-B27 transgenic) rats, an animal model of spondyloarthritis, and correlated with disease susceptibility. In this study, we demonstrate that the unfolded protein response (UPR) is activated in macrophages derived from the bone marrow of HLA-B27 transgenic rats with inflammatory disease. Microarray analysis of these cells also reveals an IFN response signature. In contrast, macrophages derived from premorbid rats do not exhibit a strong UPR or evidence of IFN exposure. Activation of macrophages from premorbid HLA-B27 transgenic rats with IFN-γ increases HLA-B27 expression and leads to UPR induction, while no UPR is seen in cells from nondisease-prone HLA-B7 transgenic or wild-type (nontransgenic) animals. This is the first demonstration, to our knowledge, that HLA-B27 misfolding is associated with ER stress that results in activation of the UPR. These observations link HLA-B27 expression with biological effects that are independent of immunological recognition, but nevertheless may play an important role in the pathogenesis of inflammatory diseases associated with this MHC class I allele.
Joel D. Taurog - One of the best experts on this subject based on the ideXlab platform.
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the human leukocyte antigen hla b27 peptidome in vivo in spondyloarthritis susceptible hla b27 transgenic rats and the effect of erap1 deletion
Molecular & Cellular Proteomics, 2017Co-Authors: Eilon Barnea, Tri M Tran, Nimman Satumtira, Martha L Dorris, Robert A. Colbert, Robert E Hammer, Dganit Melamed Kadosh, Yael Haimovich, Mylinh T Nguyen, Joel D. TaurogAbstract:HLA-B27 is a class I major histocompatibility (MHC-I) allele that confers susceptibility to the rheumatic disease ankylosing spondylitis (AS) by an unknown mechanism. ERAP1 is an aminopeptidase that trims peptides in the endoplasmic reticulum for binding to MHC-I molecules. ERAP1 shows genetic epistasis with HLA-B27 in conferring susceptibility to AS. Male HLA-B27 transgenic rats develop arthritis and serve as an animal model of AS, whereas female B27 transgenic rats remain healthy. We used large scale quantitative mass spectrometry to identify over 15,000 unique HLA-B27 peptide ligands, isolated after immunoaffinity purification of the B27 molecules from the spleens of HLA-B27 transgenic rats. Heterozygous deletion of Erap1, which reduced the Erap1 level to less than half, had no qualitative or quantitative effects on the B27 peptidome. Homozygous deletion of Erap1 affected approximately one-third of the B27 peptidome but left most of the B27 peptidome unchanged, suggesting the possibility that some of the HLA-B27 immunopeptidome is not processed in the presence of Erap1. Deletion of Erap1 was permissive for the AS-like phenotype, increased mean peptide length and increased the frequency of C-terminal hydrophobic residues and of N-terminal Ala, Ser, or Lys. The presence of Erap1 increased the frequency of C-terminal Lys and Arg, of Glu and Asp at intermediate residues, and of N-terminal Gly. Several peptides of potential interest in AS pathogenesis, previously identified in human cell lines, were isolated. However, rats susceptible to arthritis had B27 peptidomes similar to those of non-susceptible rats, and no peptides were found to be uniquely associated with arthritis. Whether specific B27-bound peptides are required for AS pathogenesis remains to be determined. Data are available via ProteomeXchange with identifier PXD005502.
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additional human β2 microglobulin curbs hla b27 misfolding and promotes arthritis and spondylitis without colitis in male hla b27 transgenic rats
Arthritis & Rheumatism, 2006Co-Authors: Tri M Tran, Nimman Satumtira, Martha L Dorris, James A Richardson, Robert E Hammer, Jie Shang, Joel D. TaurogAbstract:Objective Ankylosing spondylitis and related spondylarthritides are associated with HLA–B27, and also with intestinal inflammation, by unknown mechanisms. The folded HLA–B27 molecule is a trimer of heavy chain, β2-microglobulin (β2m), and short peptide. However, B27 heavy chain has an unusual propensity to misfold and trigger the unfolded protein response (UPR). This study was initiated to test the hypothesis that B27 misfolding plays a role in the pathogenesis of spondylarthritis. Methods Rats with high transgene copy numbers of HLA–B27 heavy chain together with human β2m (Huβ2m) spontaneously develop colitis, peripheral arthritis, and occasional spondylitis, and those with lower transgene copy numbers remain healthy. We crossed disease-prone and healthy HLA–B27/Huβ2m–transgenic rat lines with a healthy line, 283-2, carrying only the Huβ2m transgene. HLA–B27 assembly was assessed by pulse-chase analysis of B27 molecules, and UPR triggering was assessed by measuring BiP/Grp78 messenger RNA (mRNA) in splenic concanavalin A blasts. Surface expression of B27 and Huβ2m was determined by flow cytometry. Disease manifestations were identified by clinical observation, histology, and measurement of cytokine mRNA. Results The extra Huβ2m from the 283-2 line significantly reduced B27 misfolding and UPR triggering. Unexpectedly, however, F1 male offspring of the healthy 21-3 line crossed with the 283-2 line showed a high prevalence, severity, and duration of arthritis and spondylitis, in the absence of colitis. The arthropathy showed many features characteristic of human spondylarthritis. Conclusion These results suggest that B27 misfolding is associated with intestinal inflammation, but that neither B27 misfolding nor intestinal inflammation is critical to the development of B27-associated arthropathy.
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hla b27 misfolding in transgenic rats is associated with activation of the unfolded protein response
Journal of Immunology, 2005Co-Authors: Matthew Turner, Joel D. Taurog, Robert A. Colbert, Dawn P Sowders, Monica L Delay, Rajashree Mohapatra, Shuzhen Bai, Judith A Smith, Jaclyn R BrandewieAbstract:The mechanism by which the MHC class I allele, HLA-B27, contributes to spondyloarthritis pathogenesis is unknown. In contrast to other alleles that have been examined, HLA-B27 has a tendency to form high m.w. disulfide-linked H chain complexes in the endoplasmic reticulum (ER), bind the ER chaperone BiP/Grp78, and undergo ER-associated degradation. These aberrant characteristics have provided biochemical evidence that HLA-B27 is prone to misfold. Recently, similar biochemical characteristics of HLA-B27 were reported in cells from HLA-B27/human β 2 -microglobulin transgenic (HLA-B27 transgenic) rats, an animal model of spondyloarthritis, and correlated with disease susceptibility. In this study, we demonstrate that the unfolded protein response (UPR) is activated in macrophages derived from the bone marrow of HLA-B27 transgenic rats with inflammatory disease. Microarray analysis of these cells also reveals an IFN response signature. In contrast, macrophages derived from premorbid rats do not exhibit a strong UPR or evidence of IFN exposure. Activation of macrophages from premorbid HLA-B27 transgenic rats with IFN-γ increases HLA-B27 expression and leads to UPR induction, while no UPR is seen in cells from nondisease-prone HLA-B7 transgenic or wild-type (nontransgenic) animals. This is the first demonstration, to our knowledge, that HLA-B27 misfolding is associated with ER stress that results in activation of the UPR. These observations link HLA-B27 expression with biological effects that are independent of immunological recognition, but nevertheless may play an important role in the pathogenesis of inflammatory diseases associated with this MHC class I allele.
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hla b27 in transgenic rats forms disulfide linked heavy chain oligomers and multimers that bind to the chaperone bip
Journal of Immunology, 2004Co-Authors: Tri M Tran, Nimman Satumtira, Martha L Dorris, Eiichi Furuta, Andrew Z Wang, Joel D. TaurogAbstract:To test the hypothesis that HLA-B27 predisposes to disease by forming disulfide-linked homodimers, we examined rats transgenic for HLA-B27, mutant Cys67Ser HLA-B27, or HLA-B7. In splenic Con A blasts from high transgene copy B27 lines that develop inflammatory disease, the anti-H chain mAb HC10 precipitated four bands of molecular mass 78–105 kDa and additional higher molecular mass material, seen by nonreducing SDS-PAGE. Upon reduction, all except one 78-kDa band resolved to 44 kDa, the size of the H chain monomer. The 78-kDa band was found to be BiP/Grp78, and the other high molecular mass material was identified as B27 H chain. Analysis of a disease-resistant low copy B27 line showed qualitatively similar high molecular mass bands that were less abundant relative to H chain monomer. Disease-prone rats with a Cys67Ser B27 mutant showed B27 H chain bands at 95 and 115 kDa and a BiP band at 78 kDa, whereas only scant high molecular mass bands were found in cells from control HLA-B7 rats. 125I-surface labeled B27 oligomers were immunoprecipitated with HC10, but not with a mAb to folded B27-β2-microglobulin-peptide complexes. Immunoprecipitation of BiP with anti-BiP Abs coprecipitated B27 H chain multimers. Folding and maturation of B27 were slow compared with B7. These data indicate that disulfide-linked intracellular H chain complexes are more prone to form and bind BiP in disease-prone wild-type B27 and B27-C67S rats than in disease-resistant HLA-B7 rats. The data support the hypothesis that accumulation of misfolded B27 participates in the pathogenesis of B27-associated disease.
S Kollnberger - One of the best experts on this subject based on the ideXlab platform.
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the leukocyte immunoglobulin like receptor family member lilrb5 binds to hla class i heavy chains
PLOS ONE, 2015Co-Authors: Zhiyong Zhang, Hiroko Hatano, J Shaw, Marloes Olde Nordkamp, Guosheng Jiang, S KollnbergerAbstract:Objective The leukocyte immunoglobulin-like receptor (LILR) family includes inhibitory and stimulatory members which bind to classical and non-classical HLA-class I. The ligands for many LILR including LILRB5 have not yet been identified. Methods We generated C-terminal eGFP and N-terminal FLAG-tagged fusion constructs for monitoring LILR expression. We screened for LILR binding to HLA-class I by tetramer staining of 293T cells transfected with LILRA1, A4, A5 A6 and LILRB2 and LILRB5. We also studied HLA class I tetramer binding to LILRB5 on peripheral monocyte cells. LILRB5 binding to HLA-class I heavy chains was confirmed by co-immunoprecipitation. Results HLA-B27 (B27) free heavy chain (FHC) dimer but not other HLA-class I stained LILRB5-transfected 293T cells. B27 dimer binding to LILRB5 was blocked with the class I heavy chain antibody HC10 and anti-LILRB5 antisera. B27 dimers also bound to LILRB5 on peripheral monocytes. HLA-B7 and B27 heavy chains co-immunoprecipitated with LILRB5 in transduced B and rat basophil RBL cell lines. Conclusions Our findings show that class I free heavy chains are ligands for LILRB5. The unique binding specificity of LILRB5 for HLA-class I heavy chains probably results from differences in the D1 and D2 immunoglobulin-like binding domains which are distinct from other LILR which bind to β2m-associated HLA-class I.
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The Leukocyte Immunoglobulin-Like Receptor Family Member LILRB5 Binds to HLA-Class I Heavy Chains - Fig 2
2015Co-Authors: Zhiyong Zhang, Hiroko Hatano, J Shaw, Marloes Olde Nordkamp, Guosheng Jiang, S KollnbergerAbstract:LILRB5 binds specifically to HLA-B27 free heavy chain dimers but does not bind to β2m and peptide associated HLA-A3, HLA-B7 and HLA-B27 heterodimers A. FACS staining of 293 T cells transfected with eGFP and FLAG-tagged constructs of LILRB2 and LILRB5 and stained with Extravidin-PE, orExtravidin-PE conjugated tetramers of HLA-A3, HLA-B7, and HLA-B27 heterodimer or HLA-B27 free heavy chain (FHC) dimers. FACS plots show PE fluorescence from tetramer or Extravidin staining plotted against eGFP expression of each of the fusion experiments. Representative FACS stain from 1 of 3 independent experiments B. FACS staining of LILRB5 transfected 293T cells with HLA-B27 FHC dimer tetramer or Extravidin PE with or without isotype control antibody (IgG2a) or free heavy chain antibody HC10 as indicated. FACS plots show PE fluorescence from tetramer or Extravidin staining plotted against eGFP expression of each of the fusion experiments. Representative FACS stain from 1 of 3 independent experiments.
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inhibiting hla b27 homodimer driven immune cell inflammation in spondylarthritis
Arthritis & Rheumatism, 2012Co-Authors: Sravan Payeli, Osiris Marroquin Belaunzaran, Sascha Kleber, S Kollnberger, Markus Thiel, K Mchugh, Joanna L Giles, J Shaw, A Ridley, Isabel WongbaezaAbstract:Objective Spondylarthritides (SpA), including ankylosing spondylitis (AS), are common inflammatory rheumatic diseases that are strongly associated with positivity for the HLA class I allotype B27. HLA–B27 normally forms complexes with β2-microglobulin (β2m) and peptide to form heterotrimers. However, an unusual characteristic of HLA–B27 is its ability to form β2m-free heavy chain homodimers (HLA–B272), which, unlike classic HLA–B27, bind to killer cell immunoglobulin-like receptor 3DL2 (KIR-3DL2). Binding of HLA–B272 to KIR-3DL2–positive CD4+ T and natural killer (NK) cells stimulates cell survival and modulates cytokine production. This study was undertaken to produce an antibody to HLA–B272 in order to confirm its expression in SpA and to inhibit its proinflammatory properties. Methods We generated monoclonal antibodies by screening a human phage display library positively against B272 and negatively against B27 heterotrimers. Specificity was tested by enzyme-linked immunosorbent assay (ELISA), surface plasmon resonance (SPR) assay, and fluorescence-activated cell sorting (FACS) analysis of B272-expressing cell lines and peripheral blood mononuclear cells (PBMCs) and synovial fluid mononuclear cells (SFMCs) from patients with SpA. Functional inhibition of KIR-3DL2–B272 interactions was tested using cell lines and PBMCs from patients with SpA. Results Monoclonal antibody HD6 specifically recognized recombinant HLA–B272 by ELISA and by SPR assay. HD6 bound to cell lines expressing B272. FACS revealed binding of HD6 to PBMCs and SFMCs from patients with AS but not from controls. HD6 inhibited both the binding of HLA–B272 to KIR-3DL2 and the survival and proliferation of KIR-3DL2–positive NK cells. Finally, HD6 inhibited production of the proinflammatory disease–associated cytokine interleukin-17 by PBMCs from patients with AS. Conclusion These results demonstrate that antibody HD6 has potential for use in both the investigation and the treatment of AS and other B27-associated spondylarthritides.
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th17 cells expressing kir3dl2 and responsive to hla b27 homodimers are increased in ankylosing spondylitis
Journal of Immunology, 2011Co-Authors: P Bowness, Andrew J Mcmichael, J Shaw, A Ridley, Isabel Wongbaeza, A T Chan, Myles Fleming, Fraser Cummings, S KollnbergerAbstract:CD4 Th cells producing the proinflammatory cytokine IL-17 (Th17) have been implicated in a number of inflammatory arthritides including the spondyloarthritides. Th17 development is promoted by IL-23. Ankylosing spondylitis, the most common spondyloarthritis (SpA), is genetically associated with both HLA-B27 (B27) and IL-23R polymorphisms; however, the link remains unexplained. We have previously shown that B27 can form H chain dimers (termed B272), which, unlike classical HLA-B27, bind the killer-cell Ig-like receptor KIR3DL2. In this article, we show that B272-expressing APCs stimulate the survival, proliferation, and IL-17 production of KIR3DL2+ CD4 T cells. KIR3DL2+ CD4 T cells are expanded and enriched for IL-17 production in the blood and synovial fluid of patients with SpA. Despite KIR3DL2+ cells comprising a mean of just 15% of CD4 T in the peripheral blood of SpA patients, this subset accounted for 70% of the observed increase in Th17 numbers in SpA patients compared with control subjects. TCR-stimulated peripheral blood KIR3DL2+ CD4 T cell lines from SpA patients secreted 4-fold more IL-17 than KIR3DL2+ lines from controls or KIR3DL2− CD4 T cells. Strikingly, KIR3DL2+ CD4 T cells account for the majority of peripheral blood CD4 T cell IL-23R expression and produce more IL-17 in the presence of IL-23. Our findings link HLA-B27 with IL-17 production and suggest new therapeutic strategies in ankylosing spondylitis/SpA.
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interaction of hla b27 homodimers with kir3dl1 and kir3dl2 unlike hla b27 heterotrimers is independent of the sequence of bound peptide
European Journal of Immunology, 2007Co-Authors: S Kollnberger, Antoni Chan, Meiyi Sun, L Chen, Cynthia Wright, Kati Di Gleria, Andrew J Mcmichael, P BownessAbstract:HLA-B27 can form beta-2 microglobulin (β2m)-associated heterotrimers (HLA-B27) and β2m-free homodimers (B272). Here, we study the role of complexed peptide in the interaction of these forms of B27 with the killer cell immunoglobulin (Ig)-like receptors KIR3DL1 and KIR3DL2 and with Ig-like transcripts LILRB1 and LILRB2. HLA-B27 tetramers complexed with three of five different naturally processed self peptides and three of seven pathogen-derived epitopes bound to KIR3DL1-expressing transfectants and NK cells. Heterotrimeric complexes containing peptides with charged amino acids at position 8 did not bind to KIR3DL1; however, studies with analogue peptides demonstrated that these are not the only peptide residues involved in binding. KIR3DL1 ligation by HLA-B27 inhibited NK cell IFN-γ production in a peptide-dependent fashion. B27 but not HLA-A2, B7 or B57 heavy chains formed homodimers in the presence of peptide epitopes. B272 bound to KIR3DL1, KIR3DL2 and LILRB2 but not LILRB1. KIR3DL2 ligation by B272 inhibited NK and T cell IFN-γ production. By contrast with HLA heterotrimers, B272 binding to KIR did not depend on the sequence of the bound peptide. Differences in KIR binding to classical HLA and B272 could be involved in the pathogenesis of spondyloarthritis.
Andrew J Mcmichael - One of the best experts on this subject based on the ideXlab platform.
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th17 cells expressing kir3dl2 and responsive to hla b27 homodimers are increased in ankylosing spondylitis
Journal of Immunology, 2011Co-Authors: P Bowness, Andrew J Mcmichael, J Shaw, A Ridley, Isabel Wongbaeza, A T Chan, Myles Fleming, Fraser Cummings, S KollnbergerAbstract:CD4 Th cells producing the proinflammatory cytokine IL-17 (Th17) have been implicated in a number of inflammatory arthritides including the spondyloarthritides. Th17 development is promoted by IL-23. Ankylosing spondylitis, the most common spondyloarthritis (SpA), is genetically associated with both HLA-B27 (B27) and IL-23R polymorphisms; however, the link remains unexplained. We have previously shown that B27 can form H chain dimers (termed B272), which, unlike classical HLA-B27, bind the killer-cell Ig-like receptor KIR3DL2. In this article, we show that B272-expressing APCs stimulate the survival, proliferation, and IL-17 production of KIR3DL2+ CD4 T cells. KIR3DL2+ CD4 T cells are expanded and enriched for IL-17 production in the blood and synovial fluid of patients with SpA. Despite KIR3DL2+ cells comprising a mean of just 15% of CD4 T in the peripheral blood of SpA patients, this subset accounted for 70% of the observed increase in Th17 numbers in SpA patients compared with control subjects. TCR-stimulated peripheral blood KIR3DL2+ CD4 T cell lines from SpA patients secreted 4-fold more IL-17 than KIR3DL2+ lines from controls or KIR3DL2− CD4 T cells. Strikingly, KIR3DL2+ CD4 T cells account for the majority of peripheral blood CD4 T cell IL-23R expression and produce more IL-17 in the presence of IL-23. Our findings link HLA-B27 with IL-17 production and suggest new therapeutic strategies in ankylosing spondylitis/SpA.
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interaction of hla b27 homodimers with kir3dl1 and kir3dl2 unlike hla b27 heterotrimers is independent of the sequence of bound peptide
European Journal of Immunology, 2007Co-Authors: S Kollnberger, Antoni Chan, Meiyi Sun, L Chen, Cynthia Wright, Kati Di Gleria, Andrew J Mcmichael, P BownessAbstract:HLA-B27 can form beta-2 microglobulin (β2m)-associated heterotrimers (HLA-B27) and β2m-free homodimers (B272). Here, we study the role of complexed peptide in the interaction of these forms of B27 with the killer cell immunoglobulin (Ig)-like receptors KIR3DL1 and KIR3DL2 and with Ig-like transcripts LILRB1 and LILRB2. HLA-B27 tetramers complexed with three of five different naturally processed self peptides and three of seven pathogen-derived epitopes bound to KIR3DL1-expressing transfectants and NK cells. Heterotrimeric complexes containing peptides with charged amino acids at position 8 did not bind to KIR3DL1; however, studies with analogue peptides demonstrated that these are not the only peptide residues involved in binding. KIR3DL1 ligation by HLA-B27 inhibited NK cell IFN-γ production in a peptide-dependent fashion. B27 but not HLA-A2, B7 or B57 heavy chains formed homodimers in the presence of peptide epitopes. B272 bound to KIR3DL1, KIR3DL2 and LILRB2 but not LILRB1. KIR3DL2 ligation by B272 inhibited NK and T cell IFN-γ production. By contrast with HLA heterotrimers, B272 binding to KIR did not depend on the sequence of the bound peptide. Differences in KIR binding to classical HLA and B272 could be involved in the pathogenesis of spondyloarthritis.
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lymphoblastoid cells express hla b27 homodimers both intracellularly and at the cell surface following endosomal recycling
European Journal of Immunology, 2003Co-Authors: Lucy A Bird, S Kollnberger, Andrew J Mcmichael, Chen Au Peh, Tim Elliott, P BownessAbstract: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.
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cell surface expression and immune receptor recognition of hla b27 homodimers
Arthritis & Rheumatism, 2002Co-Authors: S Kollnberger, Meiyi Sun, Andrew J Mcmichael, Lucy A Bird, Christelle Retiere, Veronique M Braud, P BownessAbstract:Objective. HLA–B27 is capable of forming in vitro a heavy-chain homodimer structure lacking 2microglobulin. We undertook this study to ascertain if patients with spondylarthritis express 2-microglobulin– free HLA–B27 heavy chains in the form of homodimers and receptors for HLA–B27 homodimers. Methods. Expression of HLA–B27 heavy chains by mononuclear cells was analyzed by fluorescenceactivated cell sorter staining, Western blotting with the monoclonal antibody HC-10, and 2-dimensional isoelectric focusing. Fluorescence-labeled tetrameric complexes of HLA–B27 heavy-chain homodimers were constructed in which each dimer comprised one His-tagged heavy chain and one biotinylated heavy chain, and were used to stain patient and control mononuclear cells and transfected cell lines. Results. Patients with spondylarthritis expressed cell-surface HLA–B27 homodimers. Populations of synovial and peripheral blood monocytes, and B and T lymphocytes from patients with spondylarthritis, and controls carried receptors for HLA–B27 homodimers. Experiments with transfected cell lines demonstrated that KIR3DL1 and KIR3DL2, and immunoglobulin-like transcript 4 (ILT4), but not ILT2, are receptors for HLA–B27 homodimers. Conclusion. Patients with spondylarthritis express both HLA–B27 heavy-chain homodimers and receptors for HLA–B27 homodimers. This may be of significance with regard to disease pathogenesis.
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cutting edge hla b27 can form a novel beta 2 microglobulin free heavy chain homodimer structure
Journal of Immunology, 1999Co-Authors: Rachel L Allen, Andrew J Mcmichael, Christopher A Ocallaghan, P BownessAbstract:HLA-B27 has a striking association with inflammatory arthritis. We show that free HLA-B27 heavy chains can form a disulfide-bonded homodimer, dependent on residue Cys67 in their extracellular alpha 1 domain. Despite the absence of beta 2-microglobulin, HLA-B27 heavy chain homodimers (termed HC-B27) were stabilized by a known peptide epitope. HC-B27 complexes were recognized by the conformation-specific Ab W6/32, but not the ME1 Ab. Surface labeling and immunoprecipitation demonstrated the presence of similar W6/32-reactive free heavy chains at the surface of HLA-B27-transfected T2 cells. HC-B27 homodimer formation might explain the ability of HLA-B27 to induce spondyloarthropathy in beta 2-microglobulin-deficient mice.
