B Prakken

Epitope-specific immunotherapy of rheumatoid arthritis: clinical responsiveness occurs with immune deviation and relies on the expression of a cluster of molecules associated with T cell tolerance in a double-blind, placebo-controlled, pilot phase II trial.

OBJECTIVEnInduction of immune tolerance to maintain clinical control with a minimal drug regimen is a current research focus in rheumatoid arthritis (RA). Accordingly, we are developing a tolerization approach to dnaJP1, a peptide part of a pathogenic mechanism that contributes to autoimmune inflammation in RA. We undertook this study to test 2 hypotheses: 1) that mucosal induction of immune tolerance to dnaJP1 would lead to a qualitative change from a proinflammatory phenotype to a more tolerogenic functional phenotype, and 2) that immune deviation of responses to an inflammatory epitope might translate into clinical improvement.nnnMETHODSnOne hundred sixty patients with active RA and with immunologic reactivity to dnaJP1 were enrolled in a pilot phase II trial. They received oral doses of 25 mg of dnaJP1 or placebo daily for 6 months.nnnRESULTSnThe dnaJP1 peptide was safe and well-tolerated. In response to treatment with dnaJP1, there was a significant reduction in the percentage of T cells producing tumor necrosis factor alpha and a corresponding trend toward an increased percentage of T cells producing interleukin-10. Coexpression of a cluster of molecules (programmed death 1 and its ligands) associated with T cell regulation was also found to be a prerequisite for successful tolerization in clinical responders. Analysis of the primary efficacy end point (meeting the American College of Rheumatology 20% improvement criteria at least once on day 112, 140, or 168) showed a difference between treatment groups that became significant in post hoc analysis using generalized estimating equations. Differences in clinical responses were also found between treatment groups on day 140 and at followup. Post hoc analysis showed that the combination of dnaJP1 and hydroxychloroquine (HCQ) was superior to the combination of HCQ and placebo.nnnCONCLUSIONnTolerization to dnaJP1 leads to immune deviation and a trend toward clinical efficacy. Susceptibility to treatment relies on the coexpression of molecules that can down-regulate adaptive immunity.

CD4+ T cell responses to epitopes of self-heat shock protein 70 in patients with juvenile dermatomyositis

Background and objectives Juvenile dermatomyositis (JDM) is a childhood disease characterised by inflammation in the muscle and the skin. In several human chronic inflammatory diseases CD4 T cell responses to epitopes of heat shock proteins (hsp) have been found to play a role, both pathogenic and regulatory. In JDM hsp70 is highly up regulated in the inflamed muscle. Therefore, hsp70 could be a target in the inflammatory process in JDM. Responses to hsp70 epitopes in JDM have not yet been characterised. In HLA elution studies it has become clear that epitopes from self-hsp70 are abundantly bound to HLA. In this study the authors tested whether peripheral blood CD4 T cell responses to hsp70-epitopes can be detected and whether they are different in JDM patients compared to healthy controls (HC). Materials and methods Hsp70-epitopes were selected based on (1) literature on HLA-elution studies, (2) HLA-class II binding capacity tested with computer models and (3) in vitro responses by CD4 T cells from healthy donors. Proliferation of PBMC from healthy controls (HC, age 2–18) and JDM patients and juvenile idiopathic arthritis patients as disease controls was tested using 3H-thymidine assays and CFSE-staining. Cytokine production was tested by Luminex analysis, flow cytometry and PCR. Results The following epitopes were selected: aa 38–52, aa 161–175, aa 290–304 and aa 443–457. These peptides were immunogenic in humans, inducing proliferation or cytokine production. The proliferation of CD4 T cells towards aa 443–457 is increased in JDM. Conclusion The authors identified novel epitopes of hsp70 that are immunogenic in healthy donors and JDM and they found indications that the CD4 T cell response towards H443 is increased in JDM.

AB0202 Galectin-9 is a Robust Biomarker for Disease Activity in Juvenile Dermatomyositis and Acts as a T Cell Activator

Background Juvenile dermatomyositis (JDM) is a rare, but severe chronic systemic autoimmune disease in children, characterized by muscle weakness and a typical skin rash. Clinical evaluation of disease activity remains challenging. Recently, we identified a protein that highly correlates with disease activity in a Dutch JDM cohort: galectin-9 (gal-9). The immunobiological role of gal-9 in autoimmune diseases is still controversial. On the one hand it is known for its immunosuppressive effects by inducing apoptosis in T-helper (Th) 1 and Th17 cells and activating regulatory T cells, on the other hand it has been implicated in T cell activation and Th1 skewing. Objectives To validate the potential of gal-9 as a biomarker in JDM and investigate its immunobiological effects on T cell skewing and activation. Methods Gal-9 was measured in patients serum of an independent JDM cohort by multiplex immunoassay. For functional experiments, naive CD4 T cells were isolated and stimulated with different concentrations of gal-9, plus anti-CD3 and antigen presenting cells. To test specificity, a gal-9 blocking agent (TIM-3 fusion protein) as well as a control from the galectin family (galectin-8) were included. Flow cytometric analysis of proliferation and T cell activation markers was performed on day 3 and 5 of culture. Cytokines TNFα, IFNγ, IL-13, IL-17 and IL-10 were measured in the culture supernatants of days 3, 5 and 7 by multiplex immunoassay. Results Measurement of gal-9 in serum confirmed its high discriminative value for active disease versus remission (P=.0001; AUC 0.894; OR 9.17) even under medication, as well as a strong correlation with the clinical disease activity scores CMAS and Physicians Global VAS. On a functional level, the presence of gal-9 induced a slight but significant increase in naive CD4 T cell proliferation after 3 days of culture. The T cell activation markers CD25, CD69 and TIM-3 showed the same pattern. Gal-9 also increased production of IFNγ, TNFα and IL-10, mainly at day 7. These effects were not seen in the control conditions. Conclusions We confirmed the potential use of a very robust biomarker, galectin-9, that highly correlates with disease activity in juvenile dermatomyositis. Introduction of this biomarker into clinical practice will help to personalize treatment. Functionally, we found that gal-9 is a T cell activator, causing increased proliferation, cytokine production and expression of T cell activation markers. The high levels of circulating gal-9 in JDM patients may therefore contribute to the immunopathogenesis of JDM. References Bellutti Enders et al. Correlation of CXCL10, TNFRII, and Galectin-9 With Disease Activity in Juvenile Dermatomyositis. Arthritis Rheumatol. 2014 Aug;66(8):2281-9. Disclosure of Interest None declared

PReS-FINAL-1016: Micro vesicles as a magnifying glass; uncovering potential biomarkers in juvenile idiopathic arthritis

Juvenile idiopathic arthritis (JIA) is a common chronic inflammatory diseases in childhood. Despite remission as a result of a plethora of treatment techniques, the chronic and relapsing nature of the disease requires continuous treatment which causes adverse side effects. It is important to uncover a biomarker that can efficiently predict patient responses to therapy as well as determine if patients will progress or regress as a result of treatment. Micro vesicles are key messengers containing many immune signaling molecules including cytokines, molecules known to play a major role in JIA.

PReS-FINAL-2131: Hunting for biomarkers in juvenile dermatomyositis

Juvenile Dermatomyositis (JDM) is a systemic autoimmune disorder in which the immune system targets the microvasculature of skeletal muscles, skin and other organs, with for the most part an unknown immunopathogenesis. Moreover, evaluation of disease activity remains challenging in juvenile dermatomyositis as muscle enzyme levels and inflammatory markers, routinely used in clinics, are no reliable biomarkers in JDM, especially for monitoring the disease.

Increased stimulatory capacity of antigen presenting cells at the site of autoimmune inflammation interferes with regulatory T cell function

Background FOXP3+ regulatory T cells (Treg) are critical in maintaining self tolerance and are therefore considered important targets for the treatment of autoimmune disease. However, environmental factors at the site of autoimmune inflammation, such as enhanced costimulatory potential of antigen presenting cells (APC) and increased proinflammatory cytokine production, can negatively affect Treg function, thereby limiting effectiveness of these Treg targeted approaches. Aim Here we studied the phenotype of APC present at the site of inflammation in patients with Juvenile Idiopathic Arthritis (JIA) and investigated whether these cells can interfere with Treg mediated suppression. Methods Mononuclear cells were isolated from peripheral blood (PB) of healthy controls (HC) and from paired PB and synovial fluid (SF) of JIA patients. The phenotype of APC was analysed using flow cytometry. In vitro suppression assays were performed to study T cell activation and Treg mediated suppression in the presence of SF and PB derived APC. Results Monocytes from the site of inflammation displayed a more pro-inflammatory phenotype, with significantly increased costimulatory molecule expression, compared to monocytes from PB. In line with this pro-inflammatory phenotype, SF APC induced enhanced proliferation of effector cells and decreased suppression of effector cell proliferation in the presence of Treg. Conclusions APC from the site of inflammation have an enhanced stimulatory capacity that interferes with Treg mediated suppression. Therefore, this increased stimulatory potential should be targeted as well, in order for a Treg enhancing approach to be fully effective in the treatment of autoimmune inflammation.

METHOTREXATE DOES NOT EXERT IMMUNOMODULATORY EFFECTS ON REGULATORY T CELLS BUT ON EFFECTOR T CELLS

Background and objectives Methotrexate (MTX) is the most commonly used antirheumatic drug in juvenile idiopathic arthritis (JIA). MTX induces ‘remission on medication’ in 70% of patients and continuous ‘remission off medication’ in up to 50% of patients. MTX is unique in the realm of antirheumatic drugs since it is currently the only drug that appears capable of establishing immune tolerance in JIA. In spite of this, it is unclear which immunomodulatory mechanisms enable MTX to achieve sustained remission. The authors thus examined the immunomodulatory effects that MTX exerts on two major players in immune tolerance – regulatory (Tregs) and effector (Teffs) T cells. Materials and methods Peripheral blood mononuclear cells (PBMCs) from 25 extended oligoarticular and polyarticular JIA patients were isolated before MTX start, 3 and 6 months after MTX start. The authors analysed frequency and phenotype of Tregs by flow cytometry. The function of Tregs was evaluated in CFSE-suppression assays. Proliferation of Teffs was examined in proliferation assays in the presence of anti-CD3. Teff cytokine production was measured ex vivo with flow cytometry upon PMA-ionomycin stimulation. Results The frequency of FoxP3+ Tregs and the FoxP3 content per cell did not change upon MTX start. Suppressive capacity of Tregs appeared to be lower 6 months after MTX start compared to 3 months after MTX and prior to MTX start. In cross-over experiments, however, suppressive capacity of Tregs from all time points was equal. Teffs after MTX start proliferated significantly more in comparison to Teffs prior to MTX upon stimulation with anti-CD3. The authors observed an increase in IFN-γ and TNF-α production. Conclusions MTX does not exert immunomodulatory actions on Tregs. Instead, the authors observe changes in Teffs upon MTX start; they show that Teffs after MTX start have increased proliferation. The authors will explore the effect of MTX on Teffs further.

120* Multiplex cytokine profile detection in young children with cystic fibrosis

In young cystic fibrosis (CF) patients with mild lung disease it is unclear whether signs of a systemic inflammatory response are present. We therefore measured plasma cytokine profiles in young, clinically stable CF patients with mild lung disease. Twenty-eight different cytokines, chemokines and soluble adhesion molecules were measured in 47 CF children, ages 1.7−12.2 years, using a multiplex immunoassay. Sputum cultures were obtained on the same day as blood sampling and children were categorized as uninfected (n = 12), positive for Staphylococcus aureus and/or Haemophilus influenzae (n = 26) or positive for Pseudomonas aeruginosa (n = 9). Data were compared to those obtained from 20 healthy control children, ages 3.8−11.5 years. All CF children displayed a pro-inflammatory cytokine profile and especially interleukin (IL)-1a, IL-4, IL-12 and tumor necrosis factor-alpha (TNF-a) were significantly higher in CF children (p-values <0.001 or <0.05). Although interleukin-8 and oncostatin M (OSM) were also higher in CF children, CC-chemokines were significantly lower than in controls. In CF children bacterial sputum culture results did not influence plasma cytokine profiles, except for soluble vascular endothelial cell adhesion molecule-1 (sVCAM-1). sVCAM-1 concentrations were significantly lower in uninfected CF children compared to Pseudomonas aeruginosa positive CF children (2186.8±1207.7 versus 7170.21±2129.7, p = 0.025). Young, clinically stable CF children display a derangement of inflammatory mediator profiles. Pro-inflammatory cytokines are increased in plasma while CCchemokines are decreased. Supported by: Netherlands Organization for Health Research and Development (ZonMW).