S Kollnberger

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 β2‐microglobulin (β2m) and antigenic peptides for cell surface expression and presentation to T cells. We have previously shown that HLA‐B27 is capable of forming β2m‐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 cysteine67 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.

Interaction of HLA-B27 homodimers with KIR3DL1 and KIR3DL2, unlike HLA-B27 heterotrimers, is independent of the sequence of bound peptide.

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 positionu20048 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.

Crystal structures and KIR3DL1 recognition of three immunodominant viral peptides complexed to HLA-B*2705

We have solved the crystal structures of three HLA‐B*2705–peptide complexes with the immunodominant viral peptides: EBV EBNA3C 258–266 (RRIYDLIEL), influenza (flu) nucleoprotein NP383–391 (SRYWAIRTR), and HIV gag 264–273 (KRWIILGLNK). Long‐term non‐progression during HIV infection has been associated with presentation by HLA‐B*2705, and T cell recognition, of the highly immunodominant KRWIILGLNK peptide. The tight hydrogen‐bonding network observed between the HLA‐B*2705 B‐pocket and the peptide P2 arginine guanadinium anchor explains why mutation of this residue during HIV infection results in loss of peptide binding, immune escape and progression to AIDS. Prominent, solvent‐exposed structures within these peptides may participate in generating T cell responses to these immunodominant epitopes. In the HLA‐B*2705 complex with flu NP383–391, the amino acid side chains of residues 4, 7 and 8 are solvent‐exposed whilst in the HIV decamer, the main‐chain bulges into the solvent around P7. Thus, HLA‐B*2705 presents viral peptides in a range of conformations. Tetrameric complexes of HLA‐B*2705 with the HIV and flu but not EBV peptides bound strongly to the killer‐Ig‐like receptor (KIR)3DL1. Substitution of EBV P8 glutamate to threonine allowed recognition by KIR3DL1. In the HLA‐B*2705–EBV structure the P8 glutamate side chain is solvent‐exposed and may inhibit KIR3DL1 binding through electrostatic forces.

KIR3DL2 Binds to HLA-B27 Dimers and Free H Chains More Strongly than Other HLA Class I and Promotes the Expansion of T Cells in Ankylosing Spondylitis

The human leukocyte Ag HLA-B27 (B27) is strongly associated with the spondyloarthritides. B27 can be expressed at the cell surface of APC as both classical β2-microglobulin–associated B27 and B27 free H chain forms (FHC), including disulfide-bonded H chain homodimers (termed B272). B27 FHC forms, but not classical B27, bind to KIR3DL2. HLA-A3, which is not associated with spondyloarthritis (SpA), is also a ligand for KIR3DL2. In this study, we show that B272 and B27 FHC bind more strongly to KIR3DL2 than other HLA-class I, including HLA-A3. B272 tetramers bound KIR3DL2-transfected cells more strongly than HLA-A3. KIR3DL2Fc bound to HLA-B27–transfected cells more strongly than to cells transfected with other HLA-class I. KIR3DL2Fc pulled down multimeric, dimeric, and monomeric FHC from HLA-B27–expressing cell lines. Binding to B272 and B27 FHC stimulated greater KIR3DL2 phosphorylation than HLA-A3. B272 and B27 FHC stimulated KIR3DL2CD3ε-transduced T cell IL-2 production to a greater extent than control HLA-class I. KIR3DL2 binding to B27 inhibited NK IFN-γ secretion and promoted greater survival of KIR3DL2+ CD4 T and NK cells than binding to other HLA-class I. KIR3DL2+ T cells from B27+ SpA patients proliferated more in response to Ag presented by syngeneic APC than the same T cell subset from healthy and disease controls. Our results suggest that expansion of KIR3DL2-expressing leukocytes observed in B27+ SpA may be explained by the stronger interaction of KIR3DL2 with B27 FHC.

The role of B27 heavy chain dimer immune receptor interactions in spondyloarthritis.

HLA-B27 (B27) is strongly associated with spondyloarthopathy. The classical role of B27 is to present peptides from intracellular pathogens as a heterotrimeric complex with beta2 microglobulin for recognition by the T-cell receptor (TCR) of CD8 T-cells. In addition to heterotrimers, B27 can also be expressed as cell surface beta2-microglobulin (beta2m)-free homodimers (B27(2)). In addition to the TCR, MHC class I molecules bind to immunoregulatory receptors including members of the killer immunoglobulin-like receptor (KIR) and leukocyte immunoglobulin-like receptor (LILR) families. Rodents express the paired immunoglobulin receptor (PIR) family which are related to LILR. B27(2) but not beta2m-associated B27 binds to KIR3DL2 and rodent PIR. NK and T-cells expressing the immune receptor KIR3DL2, which interacts with B27(2), are expanded in B27 AS patients. Ligation of immune receptors by B27(2) promotes the survival of KIR-expressing leukocytes and modulates immune cytokine production. Upregulation ofB272 in spondyloarthritis and differential interaction of beta2m-associated HLA-B27 and B27(2) with immune receptors could be involved in the pathogenesis of B27-associated spondyloarthritis (AS).

Mediation of the proinflammatory cytokine response in rheumatoid arthritis and spondylarthritis by interactions between fibroblast-like synoviocytes and natural killer cells

OBJECTIVEnFibroblast-like synoviocytes (FLS) are potentially directly involved in the propagation of inflammation. We have previously shown evidence of an expanded activated population of natural killer (NK) cells in spondylarthritis (SpA) patients. In the present study, we sought to determine whether the interaction between NK cells and FLS from SpA patients results in a proinflammatory response.nnnMETHODSnAutologous NK cells and FLS were obtained from 6 patients with SpA, 4 patients with rheumatoid arthritis (RA), and 8 patients with osteoarthritis (OA). Physical interactions between NK cells and FLS were studied by time-lapse phase-contrast microscopy. Fluorescence-activated cell sorting was used to study the activation, proliferation, and survival of NK cells in contact with FLS. Cytokine and stromal factor production were measured by a multiple cytokine bead assay.nnnRESULTSnNK cells both adhered to and migrated beneath the FLS monolayer (pseudoemperipolesis). FLS from SpA and RA patients supported increased pseudoemperipolesis, activation, cytokine production, and survival of NK cells. The production of proinflammatory cytokines, including interleukin-6 (IL-6), IL-8, IL-1beta, and IL-15, was increased in cocultures of NK cells and FLS, particularly in those from RA and SpA patients. Production of interferon-gamma, RANTES, and matrix metalloproteinase 3 (MMP-3) by NK cell and FLS coculture was greatest in SpA patients. Surface expression of IL-15 on FLS was significantly increased in SpA and RA patients, but not OA patients. Blockade with an IL-15 monoclonal antibody resulted in increased apoptosis of NK cells.nnnCONCLUSIONnFLS promote the migration, activation, and survival of NK cells. The interaction of NK cells with FLS results in increased IL-15 expression by FLS and the production of proinflammatory chemokines, cytokines, and MMPs, which may contribute to joint inflammation. This response was much more marked in SpA and RA patients as compared with OA patients.

Use of HLA-B27 tetramers to identify low-frequency antigen-specific T cells in Chlamydia-triggered reactive arthritis.

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.

Expression of aberrant HLA-B27 molecules is dependent on B27 dosage and peptide supply

Objectives Cellular expression of non-classical forms of human leukocyte antigen (HLA)-B27 (NC-B27) may be involved in spondyloarthritis (SpA) pathogenesis. We used a novel B27-specific monoclonal antibody, HD6, to ask if B27 transgenic (TG) rat splenocytes express these NC-B27 molecules. We also investigated whether B27-binding peptides could affect the expression and functional immune recognition of HD6-reactive B27 molecules. Methods Splenocytes from B27-TG, B7-TG and non-transgenic rats, and HLA-B27+ cell lines were stained with monoclonal antibodies recognising classical (ME-1, HLA-ABC-m1) and non-classical (HD6, HC10) B27. Cells were further cultured in the presence of HLA-B27-binding peptides, or subjected to brief low pH treatment prior to mAb staining and/or immunoprecipitation or co-culture with KIR3DL2-CD3ε-expressing Jurkat reporter cells. Results HD6-reactive molecules were detected in the majority of adult B27-TG rat splenocyte cell subsets, increasing with age and concomitant increased B27 expression. HD6 staining was inhibited by incubation with B27-binding peptides and induced by low pH treatment. HD6 staining correlated with KIR3DL2-CD3ε-expressing Jurkat reporter cell activity. Thus, IL-2 production was decreased when B27-expressing antigen-presenting cells were preincubated with B27-binding peptides, but increased following pretreatment with low pH buffer. Conclusions Surface expression of HD6-reactive B27 molecules on B27-TG rat splenocytes is consistent with a pathogenic role for NC-B27 in SpA. Interaction of NC-B27 with innate immune receptors could be critical in SpA pathogenesis, and we show that this may be influenced by the availability and composition of the B27-binding peptide pool.

THE DETECTION OF A POTENTIALLY PATHOGENIC SUBSET OF HLA-B27 CONFORMERS AT THE CELL SURFACE IN A RAT MODEL OF SPA

Background The MHC class I antigen HLA-B27 (B27) is strongly associated with development of SpA, yet the mechanism by which B27 confers this susceptibility is unclear. B27 exists at the cell surface as fully conformed heterotrimers (B27HT) associated with peptide and b2m, but also as peptide-free heavy chain monomers (B27HC) and heavy chain homodimers (B272), which we have shown to interact with innate immune receptors in a distinct manner to B27HT. A pathogenic role for these receptor interactions has been proposed. Objectives This study aimed to analyse the expression of these potentially pathogenic aberrant forms of B27 in splenocytes isolated from B27 TG rats through the use of a novel B27-specific monoclonal antibody, HD6. Methods Splenic mononuclear cells from age-matched adult (6-9 months) B27 transgenic (TG), B7 TG, and non-transgenic (NT) rats were compared for staining with the monoclonal antibodies HD6, HC-10, HLA-ABC-m1 and an IgG1 isotype control. Staining was also compared in splenocytes isolated from young clinically healthy B27 TG rats (5-10 weeks) with those from the adult B27 TG rats. For further characterisation of the HD6 epitope, splenocytes were cultured with HLA-B27-binding or control peptides prior to FACS analysis. Results HD6 staining was consistently seen in the lymphocyte and macrophage/DC populations for adult B27 TG samples. Notably, however, no HD6 staining above background was observed in the granulocyte gate, which was also the case for HC-10. Within the lymphocyte, gate, comparable staining was seen for the CD4+ and CD8+ cell populations. HD6 staining was B27-specific, as B7 TG rat splenocytes consistently failed to stain with HD6, despite similar expression of human MHC class I molecules. Expression of folded HLA-B27 was increased in the older B27 TG rats compared to the younger B27 TG rats, consistent with previous reports, as was expression of free heavy chains. However, very little detectable HD6 staining was observed in cell subsets from the younger rats. HD6 staining was inhibited by incubation of splenocytes with B27-binding peptides. Conclusions These data demonstrate cell surface expression of HD6-reactive molecules on a number of cell subsets from HLA-B27 transgenic rats. Moreover, HD6 surface staining is strongly correlated both with the degree of B27 expression and with age. HD6 binding can be inhibited by incubation with B27-binding peptides, suggesting that it detects a population of peptide-free aberrant cell surface B27 conformers. Our data is consistent with the hypothesis that altered immune interactions, as a consequence of the presence of aberrant forms of HLA-B27 at the cell surface, contribute to SpA pathogenesis. Disclosure of Interest None Declared

TH17 CELLS EXPRESSING KIR3DL2 AND ENRICHED FOR GUT HOMING MARKERS ARE INCREASED IN ANKYLOSING SPONDYLITIS

Background T helper 17 (Th17) cells are a subset of pro-inflammatory CD4 T cells implicated in a number of inflammatory arthritides including the Spondyloarthritides (SpAs). Ankylosing Spondylitis (AS), the commonest spondyloarthropathy, is genetically associated with HLA-B27 (B27) and IL-23 receptor polymorphisms, however the link remains unexplained. We have previously shown KIR3DL2+CD4 T cells are expanded in the peripheral blood of individuals with AS. Objectives The aim of the study was to further characterize KIR3DL2+CD4 T cells and investigate whether activation induced expression of KIR3DL2 on CD4 T cells. Methods KIR3DL2+ CD4 T cell phenotype was investigated by flow cytometry. Production of cytokines by PMA/ionomycin stimulated-PBMCs was investigated by intracellular cytokine staining (ICS). Cytokine production by α-CD3/28-stimulated FACS-sorted KIR3DL2+ and KIR3DL2- CD4 T cells was investigated by multiplex bead analysis. Expression of KIR3DL2+ on CD4 T cells was investigated after SEB stimulation and cytokine production was investigated by ELISA. Results KIR3DL2+ CD4 T cells, increased in peripheral blood of HLA-B27+ SpA patients, were enriched for expression of Th17 phenotypic markers, IL-23R, CCR6 and IL-1R, and the gut-homing chemokine receptor, CCR9. KIR3DL2+ CD4 T cells from AS patients produced significantly more IL-17 than KIR3DL2- CD4 T cells. IL-17 levels significantly increased in the presence of the Th17 cytokines rIL-23 and IL-1. SEB activation increased the number of KIR3DL2+ cells and IL-17 production more in AS patients than controls. Conclusions KIR3DL2+ CD4 Th17 cells are expanded in patients with Spondyloarthritis. Expression of KIR3DL2 on CD4 T cells can be induced by activation. These cells constitute a significant proportion of peripheral blood CD4 T IL-23R-expressing cells and produce increased levels of IL-17, which is further increased by the presence of Th17 cytokines. Our findings would support the trial of new therapeutic strategies, such as anti-IL-17, in AS/SpA. Disclosure of Interest None Declared