M H Al-Mossawi

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 role of natural killer cells, gamma delta T-cells and other innate immune cells in spondyloarthritis.

Purpose of reviewNatural killer (NK) cells, gamma delta (&ggr;&dgr;) T-cells and other innate immune cells are important lymphocyte subsets able both to produce cytokines including the pro-inflammatory cytokine IL-17 and to kill cellular targets. This review describes the features of NK cells, &ggr;&dgr; T-cells and other innate immune cells, and outlines the evidence for their potential pathogenic roles in spondyloarthritis (SpA). Recent findingsNK cells and T cells both express receptors that recognize aberrantly folded human leucocyte antigen. This interaction seems to polarize towards a type 17 immunity programme which has been increasingly implicated in SpA pathology. &ggr;&dgr; T-cells have also been shown to be polarized towards a type 17 immunity programme in SpA. Gut interactions with the microbiome can influence NK and innate lymphoid immune responses in SpA and other related diseases. A newly identified population of resident lymphoid cells at the enthesis for the first time offers an explanation for the anatomical localization of SpA. SummaryNK cells, &ggr;&dgr; T-cells and other innate immune cells are capable of sharing expression of both transcription factors, including ROR&ggr;t, and cell surface receptors, such as the killer immunoglobulin-like receptors. There is increasing genetic and functional evidence that they contribute to the ROR&ggr;t-driven inflammatory type 17 immune responses, and they may link gut inflammation and joint pathology in SpA.

miR-10b-5p is a novel Th17 regulator present in Th17 cells from ankylosing spondylitis

Objective To determine the microRNA (miR) signature in ankylosing spondylitis (AS) T helper (Th)17 cells. Methods Interleukin (IL)-17A-producing CD4+ T cells from patients with AS and healthy controls were FACS-sorted for miR sequencing and qPCR validation. miR-10b function was determined by miR mimic expression followed by cytokine measurement, transcriptome analysis, qPCR and luciferase assays. Results AS Th17 cells exhibited a miR signature characterised by upregulation of miR-155-5p, miR-210-3p and miR-10b. miR-10b has not been described previously in Th17 cells and was selected for further characterisation. miR-10b is transiently induced in in vitro differentiated Th17 cells. Transcriptome, qPCR and luciferase assays suggest that MAP3K7 is targeted by miR-10b. Both miR-10b overexpression and MAP3K7 silencing inhibited production of IL-17A by both total CD4 and differentiating Th17 cells. Conclusions AS Th17 cells have a specific miR signature and upregulate miR-10b in vitro. Our data suggest that miR-10b is upregulated by proinflammatory cytokines and may act as a feedback loop to suppress IL-17A by targeting MAP3K7. miR-10b is a potential therapeutic candidate to suppress pathogenic Th17 cell function in patients with AS.

Unique transcriptome signatures and GM-CSF expression in lymphocytes from patients with spondyloarthritis

Spondyloarthritis encompasses a group of common inflammatory diseases thought to be driven by IL-17A-secreting type-17 lymphocytes. Here we show increased numbers of GM-CSF-producing CD4 and CD8 lymphocytes in the blood and joints of patients with spondyloarthritis, and increased numbers of IL-17A+GM-CSF+ double-producing CD4, CD8, γδ and NK cells. GM-CSF production in CD4 T cells occurs both independently and in combination with classical Th1 and Th17 cytokines. Type 3 innate lymphoid cells producing predominantly GM-CSF are expanded in synovial tissues from patients with spondyloarthritis. GM-CSF+CD4+ cells, isolated using a triple cytokine capture approach, have a specific transcriptional signature. Both GM-CSF+ and IL-17A+GM-CSF+ double-producing CD4 T cells express increased levels of GPR65, a proton-sensing receptor associated with spondyloarthritis in genome-wide association studies and pathogenicity in murine inflammatory disease models. Silencing GPR65 in primary CD4 T cells reduces GM-CSF production. GM-CSF and GPR65 may thus serve as targets for therapeutic intervention of spondyloarthritis.Spondyloarthritis is an inflammatory disease with Th17 cells implicated in the pathogenesis. Here the authors show that patients with spondyloarthritis have increased numbers of GM-CSF-secreting blood and synovial lymphocytes, Th17 or not, that carry a unique transcriptional profile including enhanced GPR65 expression.

Role of lymphocytes producing GM-CSF in human spondyloarthritis

Abstract Background The spondyloarthritides (SpA) are immune-driven inflammatory diseases in which T lymphocytes are thought to have a major pathogenic role. Lymphocytes producing granulocyte-macrophage colony-stimulating factor (GM-CSF) have been shown to be especially pathogenic in murine models of SpA. This study aimed to identify and characterise the role of GM-CSF-producing lymphocytes in human SpA. Methods Peripheral blood and joint-derived mononuclear cells from adults with SpA and controls (healthy adults and adults with the inflammatory joint disease rheumatoid arthritis) were studied with flow cytometry and time of flight mass cytometry (CyTOF). The role of GM-CSF was also investigated in joint-derived innate lymphoid cells. GM-CSF-producing CD4 lymphocytes were isolated by cytokine capture, and RNA sequencing transcriptomic analysis was used to compare GM-CSF lymphocytes with T helper (Th) 1 and Th17 cells. Findings An expansion of GM-CSF-producing CD4 and CD8 T cells was noted in the blood and joints of patients with SpA. Flow cytometry and CyTOF showed that CD4 T cells were the main producers of GM-CSF. GM-CSF production occurred both independently and in combination with classic Th1 and Th17 cytokines, with increased numbers of interleukin 17A+GM-CSF+ double-producing CD4, CD8, and natural killer cells. Expansion of type 3 innate lymphoid cells in SpA also occurred, and GM-CSF was abundantly produced by these cells. Transcriptional analysis of GM-CSF+ cells isolated with a novel triple cytokine capture approach suggested that human GM-CSF+ CD4 T cells represent an effector population distinct from Th1 and Th17 cells. Interpretation GM-CSF is a key effector cytokine in human SpA and may be a valid target for therapeutic intervention. Anti-GM-CSF monoclonal antibodies are currently in phase 3 trials for rheumatoid arthritis and have been shown to be safe in patients. These data would support the use of these antibodies in SpA. Funding Wellcome Trust clinical PhD award (to MHA-M).

COMPARISON OF IN VITRO EFFECTS OF KINASE AND EPIGENETIC INHIBITORS ON TH17 RESPONSES IN INFLAMMATORY ARTHRITIS

Background Th17 cells producing the proinflammatory cytokines IL-17A and IL-22 mediate multiple autoimmune conditions including inflammatory arthritides, and among these ankylosing spondylitis (AS) (1, 2). The role of Th17 cells in AS pathogenesis is supported by the efficacy of an anti-IL-17A antibody, secukinumab, in a clinical trial (3). JAK inhibitors like tofacitinib act on Th17 cells in rheumatoid arthritis (RA) (4). In additon to this a bromodomain inhibitor, JQ1, inhibits Th17 polarization in vitro and ameliorates disease in a murine model of arthritis (1). Objectives Against the background of restricted therapeutic options besides NSAID and anti-TNF therapy in AS we representatively evaluated the in vitro actions of tofacitinib, a JAK1/3 inhibitor, and JQ1, a BET bromodomain inhibitor, on Th17 responses of patient-derived CD4+ T cells. Methods Peripheral blood mononuclear cells were isolated from patients with AS, psoriatic arthritis (PSA) and RA and from healthy controls (HC). CD4+ T cells were negatively selected with magnetic beads and cultured in Th17-polarizing conditions (anti-CD2/3/28 beads, recombinant IL-2, IL-1β, IL-6 and IL-23). Cells were treated from day 0 on with either tofacitinib or JQ1. After 3 days IL-17A secretion was measured in the supernatant by ELISA and cells were analyzed for cytotoxic effects with a WST-1 assay. On day 6 of culture cells were stained for intracellular cytokines (IL-17A, IFNγ, IL-22, TNFα) and analyzed by flow cytometry. At the same time point cellular proliferation was assessed by a CFSE assay. Results Both inhibitors effectively reduced IL-17A secretion from Th17-polarized CD4+ T cells. JQ1 also decreased percentages of total IL-17A+ T cells in all diseases and controls, whereas tofacitinib increased percentages of total IL-17A+ T cells in HC, AS and PSA. In addition a reduction of total IFNγ+ T cells in HC, AS and PSA, and no changes of total IL-22+ or TNFα+ T cells in all groups were observed upon treatment with JQ1. Tofacitinib elevated total IL-22+ T cells in HC, AS and PSA. An inhibitory effect of JQ1 on IL-17A+/IFNγ+ T cells was only obvious in AS. IL-17A+/IL-22+ T cells were not effected by either of the inhibitors. However JQ1 and tofacitinib reduced viability of Th17-polarized CD4+ T cells by about 50% in HC and AS. The effect on proliferation was less remarkable for JQ1 compared to tofacitinib in both groups. Conclusions Our results indicate that the BET bromodomain inhibitor JQ1 (compared to the JAK1/3 inhibitor tofacitinib) significantly inhibits Th17 responses in CD4+ T cells derived from patients with AS, PSA and RA. This encourages further investigation of JQ1 in animal models and the search for more specific derivates of this compound. References Mele, D. et al. BET bromodomain inhibition suppresses Th17-mediated pathology. J. Exp. Med. 210, 2181-2190 (11) Bowness, P. et al. Th17 cells expressing KIR3DL2+ and responsive to HLA-B27 homodimers are increased in ankylosing spondylitis. J. Immunol. 186, 2672-2680 (4) Baeten, D. et al. Anti-interleukin-17A monoclonal antibody secukinumab in treatment of ankylosing spondylitis: a randomised, double-blind, placebo-controlled trial. The Lancet 382, 1705–1713 (23) Maeshima, K. et al. The JAK inhibitor tofacitinib regulates synovitis through inhibition of interferon-γ and interleukin-17 production by human CD4+ T cells. Arthritis Rheum. 64, 1790-1798 (6) Acknowledgements JQ1 was generously provided by Knapp S. and Fedorov O. (SGC, University of Oxford) and funding for Hammitzsch A. by the DFG. Disclosure of Interest A. Hammitzsch Grant/research support: DFG grant, HA 7021/1-1, J. de Wit Grant/research support: Merck, A. Ridley: None declared, M. H. Al-Mossawi Grant/research support: Merck, P. Bowness Grant/research support: Merck DOI 10.1136/annrheumdis-2014-eular.2963

OP0286 Genotypic effects of ankylosing spondylitis associated il7r polymorphisms are mediated through monocytes in inflammation

Background Interleukin 7 (IL-7) plays a key role in T cell survival and proliferation. Both cell-surface expressed and soluble forms of the IL-7 receptor (sIL7R) are recognised. sIL7R has been shown to prolong IL-7 activity in inflammation(.1 IL7R polymorphisms are associated with multiple inflammatory diseases including ankylosing spondylitis(AS). Higher levels of circulating sIL7R are present in those carrying the risk variant but the cellular sources of soluble IL7R are unclear. Expression quantitative trait loci (eQTL) studies have shown genetically regulated IL7R gene upregulation in monocytes after innate immune stimuli(.2 Objectives This study aims to characterise the genotypic effects of IL7R polymorphism on monocyte protein surface expression and sIL7R release. Methods Monocyte cell surface IL7R expression was measured by flow cytometry in the presence or absence of LPS or TNF in a cohort of volunteers recruited from the Oxford biobank. Soluble IL7R was quantified by ELISA in purified monocyte cultures stimulated with LPS. RNA sequencing was performed for 8 paired samples of control and recombinant IL-7 exposed stimulated monocytes. Results Surface IL7R expression was induced after 24 hours of LPS stimulation both in isolated monocytes (n=84, p=<8.9e-19) and CD14 +cells in whole PBMC cultures (n=103, p=<3.9e-31). We find the key genotypic regulator of this response to be rs6897932, previously associated with AS predisposition, both in isolated monocytes (n=85, p=9e-7) and CD14 +cells in whole PBMC cultures (n=103, p=9.4e-5). There was no genotypic effect seen in unstimulated monocytes. Notably IL7R positivity of CD4+, CD8 +and CD56+cells measured within the PBMC culture both before or after LPS stimulation showed no association with rs6897932. The addition of anti-TNF (Infliximab) abrogated the genotypic effect. In a second independent cohort, genotype-specific surface IL7R induction was also observed after stimulation with recombinant human TNF (n=62, p=0.0007). In a third cohort, release of soluble IL7R was observed to be under genetic control after stimulation with LPS (n=99, p=<3.1e-11). RNA sequencing of isolated monocytes stimulated with LPS and recombinant IL-7 showed differential expression of 3240 transcripts (FDR<0.05) compared with LPS alone (n=8). Direct ex-vivo staining of monocytes from inflamed synovial fluid of SpA showed significant upregulation of IL7R compared to paired PBMC monocytes (n=4, p=<0.015). Conclusions Monocytes upregulate IL7R expression and soluble IL7R secretion after LPS treatment in a functional, genotype- and TNFalpha-dependant manner. SpA synovial monocytes express IL7R suggesting preactivation. These data draw attention to an unappreciated key myeloid role for AS risk variants at IL7R. References [1] Lundström W, et al. PNAS2013. [2] Fairfax BP, et al. Science2014. Acknowledgements BPF and PB contributed equally to this work Disclosure of Interest None declared

IDENTIFICATION AND PHENOTYPING OF INNATE LYMPHOID CELLS PRESENT IN THE DISEASED JOINTS OF PATIENTS WITH SPONDYLOARTHRITIS, RHEUMATOID ARTHRITIS AND PSORIATIC ARTHRITIS

Background and aims Innate lymphoid cells (ILCs) have been recently identified at a number of different tissues and are thought to be an important class of innate immune cells involved in the initiation of the inflammatory response during health and disease. It is not known whether these cells exist in the human joint. We set out to identify and characterise these cells in the joints of patients with inflammatory arthritis. Methods Matched synovial fluid and peripheral blood mononuclear cells from patients with spondyloarthritis (SpA) and psoriatic arthritis (PsA) were isolated and phenotyped using flow cytometry. In addition, cells from explanted orthopaedic surgical tissue samples were obtained from patients with SpA, PsA and rheumatoid arthritis (RA) and cultured in media containing interleukin-2 and interleukin-7. Intracellular cytokine staining was performed and analysed by flow cytometry. Results A population of lineage-negative (i.e. negative for CD3, CD5 CD8, CD11b, CD11c, CD14, CD19, CD20, CD34, TCR-γδ) CD45 positive, IL-7R positive cells was identified in the synovial fluid and tissue of diseased human joints. Further phenotyping showed these to be either C-KIT positive ILC3 cells or C-KIT negative ILC1 cells. CRTH2 positive ILC2 cells were not detected. Comparison of matched blood and synovial fluid cells showed ILC3 populations were enriched in the joint and expressed HLA-DR. Discussion and conclusions ILC populations (type 1 and type 3) are pre present in the synovial fluid and synovial tissue of inflamed SpA, PsA and RA joints. These cells are capable of rapid cytokine release and are potentially highly inflammatory. They may also play a role in antigen presentation through HLA-DR expression. Understanding the role of ILCs in health and disease is important for deciphering the processes taking part in early SpA pathogenesis.

IN-VITRO SUPRESSION OF TH17 RESPONSES IN INFLAMMATORY ARTHRITIS PATIENTS USING SMALL MOLECULE ROR-GAMMA-T INHIBITORS

Background Increasing evidence implicates the type 17 immune axis in the pathogenesis of inflammatory arthritidies including ankylosing spondylitis (AS), and targeting of the pathway in AS has shown benefit in early clinical trials1. Cells driving inflammation in this axis express the transcription factor retinoid-related orphan nuclear receptor gamma T (ROR-gt) and make the signature pro-inflammatory cytokine interleukin-17A (IL-17A)2. Digoxin, a small-molecule inhibitors of ROR-gt has proved successful in the treatment of mouse experimental autoimmune encephalomyelitis3 but the dose needed to achieve inhibition in human cells is over 100 fold greater than the toxic threshold. New non-digoxin small molecule ROR-gt inhibitors have now been developed and we explore their role in the human inflammatory arthritidies. Objectives To determine the effects of novel small molecule ROR-gT inhibitors on in-vitro peripheral blood derived Type 17 T cell responses of patients with inflammatory arthritis. Methods Blood samples were obtained from patients with AS, rheumatoid arthritis (RA) and psoriatic arthritis (PsA). CD4 positive type 17 cells were expanded from peripheral blood monocuclear cells (PBMCs) of patients with inflammatory arthritis using low strength anti-CD2/3/28 stimulation and recombinant IL-2 in a 7 day culture system. Two different ROR-gt inhibiting compounds (A & B) were added at the start of the culture system and their effects on multiple cytokine expression (IL-17A, IFN-gamma, IL-22, GM-CSF, TNF-alpha) was determined by flow cytometry using intracellular staining. IL-17A production was validated using ELISA. Results Both ROR-gt small molecule inhibitors resulted in a consistent decrease in the percentage of patient-derived IL-17A-producing CD4 T cells. Overall there was approximately a 50% reduction in IL-17A production in AS (n=24, p<0.0001. see fig). The inhibition of IL-17A was confirmed on ELISA. There was a similar level of inhibition observed in IL-17F production. IL-22/IL-17A double producing cells were also inhibited but there was no significant effect on IL-22 single producers (Th22 cells). The effects of these inhibitors seem to be specific to type-17 cytokines (IL-17A, IL-17F) since we did not observe significant effects on the total amounts of other measured cytokines (IFN-gamma, TNF-alpha, GM-CSF). We saw inhibition of IL-17A production in RA and PsA. ROR-gt compounds did not adversely affect cell viability or proliferation in our culture system. Figure 1. Effects of ROR-gt inhibitor (compound A) on PBMC-derived Th17 cells from AS, RA and PsA patients. Conclusions Our results demonstrate a consistent and specific inhibition of IL-17A responses from patient derived PBMCs using small molecule ROR-gt inhibitors. We believe these results provide a solid rationale for the testing of these compounds in clinical trials particularly in AS where the treatment options remain limited. References Baeten, D. et al. Anti-interleukin-17A monoclonal antibody secukinumab in treatment of ankylosing spondylitis: a randomised, double-blind, placebo-controlled trial. The Lancet 382, 1705–1713 (23). Yang, X. O. et al. T Helper 17 Lineage Differentiation Is Programmed by Orphan Nuclear Receptors RORα and RORγ. Immunity 28, 29–39 (2008). Huh, J. R. et al. Digoxin and its derivatives suppress TH17 cell differentiation by antagonizing RORγt activity. Nature 472, 486–490 (2011). Acknowledgements Inhibitor compounds and funding for research consumables was provided by Merck. Disclosure of Interest : M. H. Al-Mossawi Grant/research support: Merck, J. De Wit Grant/research support: Merck, M. Huhn Grant/research support: Merck, A. Ridley: None declared, H. Bunting: None declared, C. Arancibia Grant/research support: Merck, F. Powrie Grant/research support: Merck, P. Bowness Grant/research support: Merck DOI 10.1136/annrheumdis-2014-eular.1110