P. Blanco

T follicular helper (Tfh) cells in lupus: Activation and involvement in SLE pathogenesis

Systemic lupus erythematosus (SLE) is a chronic systemic inflammatory autoimmune disease characterized by a breakdown of tolerance to self. The autoantibodies generated in SLE are directed against nuclear components, with which they form immune complexes (ICs). ICs play key roles in organ and tissue damage, as well as in the activation of the innate and adaptive immune system during the disease course. Therefore, it is of prime importance to understand the mechanisms responsible for the development of B cells producing these pathogenic autoantibodies. There is compelling evidence that T follicular helper (Tfh) cells play a fundamental role in this process. In this review, we will summarize the current knowledge regarding the involvement of Tfh cells in SLE pathogenesis, and discuss potential strategies to target Tfh cells and/or molecules as a therapeutic modality of SLE.

Platelets Induce Thymic Stromal Lymphopoietin Production by Endothelial Cells: Contribution to Fibrosis in Human Systemic Sclerosis.

To investigate the relationship between vascular damage and fibrosis in systemic sclerosis (SSc) by testing the hypothesis that platelets contribute to skin fibrosis via the activation of human dermal microvascular endothelial cells (HDMECs) and subsequent production of profibrotic mediators.

Platelets induce thymic stromal lymphopoietin production by endothelial cells: Contribution to human systemic sclerosis fibrosis

To investigate the relationship between vascular damage and fibrosis in systemic sclerosis (SSc) by testing the hypothesis that platelets contribute to skin fibrosis via the activation of human dermal microvascular endothelial cells (HDMECs) and subsequent production of profibrotic mediators.

Repeated pulses of methyl-prednisolone with reduced doses of prednisone improve the outcome of class III, IV and V lupus nephritis: An observational comparative study of the Lupus-Cruces and lupus-Bordeaux cohorts

OBJECTIVEnTo compare the clinical course of patients with class III, IV and V lupus nephritis (LN) treated at Hospital Universitario Cruces (CC) and at Bordeaux University Hospital (BC).nnnMETHODSnThe Lupus-Cruces nephritis protocol combines pulses of 125mg of methyl-prednisolone with each fortnightly pulse of cyclophosphamide and prednisone ≤30mg/day with tapering over 12-14weeks until 2.5-5mg/day. The BC followed international lupus nephritis guidelines, combining high-dose prednisone and either mycophenolate mofetil or cyclophosphamide, followed by maintenance therapy with low dose prednisone and immunosuppressive drugs. The main outcomes were complete renal remission (CR) and glucocorticoid toxicity.nnnRESULTSn44 patients from BC and 29 from CC were included. The mean maximum prednisone dose was 42.5 (BC) vs. 21mg/day (CC), p<0.001. The average 6-month prednisone dose was 21 (BC) vs. 8.3mg/d (CC), p<0.001.The mean number of methyl-prednisolone pulses was 3 (BC) vs. 9.3 (CC), p<0.001. HCQ was used by 64% (BC) vs. 100% (CC), p<0.001. CR rates were 30% (BC) vs. 69% (CC), p=0.001, and 42% (BC) vs. 86% (CC), p<0.001, at 6 and 12months, respectively. Patients from the CC more frequently achieved CR (adjusted HR 3.8, 95%CI 2.05-7-09). The number of pulses of methyl-prednisolone were associated with CR (adjusted HR 1.09, 95%CI 1.03-1.15). Patients in the CC had a lower risk of GC-related side effects (adjusted HR 0.19, 95%CI 0.04-0.89).nnnCONCLUSIONnThe Lupus-Cruces nephritis protocol improves the outcome of LN. Repeated methyl-prednisolone pulses help reduce the dose of oral glucocorticoids and enhance clinical response.

Association of the Presence of Anti-Carbamylated Protein Antibodies in Early Arthritis With a Poorer Clinical and Radiologic Outcome: Data From the French ESPOIR Cohort

To assess the prevalence of anti–carbamylated protein (anti‐CarP) antibodies in a French cohort of patients with early arthritis and to investigate their association with clinical features, final diagnosis, prognosis, and comorbidities.

Expansion of myelin autoreactive CD8+ T lymphocytes in patients with neuropsychiatric systemic lupus erythematosus

Objectives Delineation of mechanisms underlying neuropsychiatric systemic lupus erythematosus (NPSLE) and determination of biological markers could guide treatment choice. A study was undertaken to analyse the potential role of activated CD8+ T cells in NPSLE as previously reported in SLE nephritis. Methods Flow cytometric immunophenotyping of blood lymphocytes was performed in 30 patients with NPSLE and 36 healthy individuals. The antigenic specificity of CD8+ T cells was studied using HLA-A0201 tetramers loaded with several myelin-derived peptides. The intracellular level of interferon γ (IFNγ) produced by activated CD8+ T cells was determined by flow cytometry. Results A large increase in circulating activated CD8+ T lymphocytes expressing surface HLA-DR was found in patients with NPSLE without antiphospholipid syndrome (APS) (n=18) compared with patients with APS (n=12) or healthy controls (n=36). IFNγ-secreting myelin-specific CD8+ T cells were detected exclusively in the blood of patients with NPSLE without APS but with white matter lesions. Conclusions These data strongly support the existence of a new immune effector mechanism responsible for CNS involvement in patients with NPSLE and suggest that analysing HLA-DR expression combined with myelin-specific tetramer staining on CD8+ T lymphocytes may be a valuable additional tool for the monitoring of these patients.

Anti-carbamylated protein antibodies presence in early arthritis with a poorer clinical and radiological outcome: data from the French ESPOIR cohort

To assess the prevalence of anti–carbamylated protein (anti‐CarP) antibodies in a French cohort of patients with early arthritis and to investigate their association with clinical features, final diagnosis, prognosis, and comorbidities.

SAT0191 Platelets Induce Thymic Stromal Lymphopoietin Production by Endothelial Cells: Contribution To Human Systemic Sclerosis Fibrosis

Background Systemic sclerosis (SSc) is a complex systemic autoimmune disease characterized by microvascular dysfunction, immune activation and fibrosis affecting the skin and internal organs (1). Improved characterization of the fibrotic mechanisms involved in SSc is a keystone to developing biomarkers and effective disease-modifying therapies. The activation of endothelial cells (ECs) appears very early in the course of the disease, suggesting that chronic fibroblast activation may be at least partly the result of endothelial dysfunction (1,2). However, the ability of the platelet/EC interaction to induce the secretion of factors directly involved in fibroblast activation and subsequent fibrosis in SSc remains uncertain. Objectives The objective was to provide evidence of a new pathogenic loop implicating activated platelets, microvascular ECs and ECM production in SSc. Methods A total of 203 SSc patients and 30 healthy donors (HDs) were prospectively enrolled between March 2012 and January 2015 at the University Hospital of Bordeaux. Immunohistochemistry and immunofluorescence analysis were performed on skin biopsy specimens from 18 SSc patients and 5 HDs. Serum TSLP levels were measured (ELISA) in the entire cohort. Human dermal microvascular ECs and fibroblasts were purified from normal and SSc patients skin biopsies. Extracellular matrix production by cultured fibroblasts was assessed by RT-qPCR. Results Serum TSLP levels were significantly increased in SSc patients compared to HDs (p=0.0005) and were associated with a higher frequency of vascular damage (p=0.02). The proportion of dermal TSLP-positive cells was increased in the skin biopsies of SSc patients compared to HDs (p=0.0008) and was correlated with skin fibrosis (modified Rodnan skin score) (R=0.059, p=0.01). In SSc dermis, TSLP was mainly expressed by CD31-positive ECs. In vitro, activated platelets induced TSLP production by dermal microvascular ECs in an IL1β-dependent manner. Recombinant TSLP in normal fibroblasts reproducibly induced a pro-fibrotic profile as indicated by a significant increase in the collagen/collagenase ratio (p=0.03). Interestingly, this property was shared by fibroblasts purified from non-fibrotic TSLP-negative SSc skin, but lost with fibroblasts purified from TSLP-positive fibrotic skin. Conclusions Taken together, these results identify dermal microvascular ECs as new contributors to TSLP production in SSc and suggest a potential mechanism by which platelets may profoundly affect the fibrotic process in SSc. References Gabrielli A, et al., N Engl J Med 2009. Pattanaik D, et al., Front Immunol 2015. Disclosure of Interest None declared

OP0046 Decreased Circulating ILC2 Levels Correlate with Skin Fibrosis in Human Systemic Sclerosis and Are Associated with A Skewing of The ILC1/ILC2 Balance in The Skin

Background Interleukin (IL)13 is considered as a key downstream mediator in the development of Systemic sclerosis (SSc) fibrosis1. The recently described type 2 innate lymphoid cells (ILC) produce higher amounts of IL13 than Th2 cells under certain stimulatory conditions2. Our recent work show that endothelial cells are an important source of thymic stromal lymphopoietin (TSLP) in SSc3, which is an inducer of IL13 production by ILC2. Altogether, we assume that ILC2 could have a role in the promotion of SSc fibrosis. Objectives To analyse ILC populations in whole blood and skin of SSc patients and heatlhy donors (HD) and to correlate to clinical parameters. Methods 25 SSc and 15 HD were prospectively enrolled between September 2015 and January 2016 in the University Hospital of Bordeaux. Skin biopsies were performed in fibrotic skin for 2 SSc patients and in pieces of brachioplasty for HD. All patients signed informed consent. Analysis were done on fresh blood after Red Blood lysis and on cells from skin biopsies digested with liberase and DNase. ILC were measured by flow cytometry using Beckman Coulter and Miltenyi Biotech kits respectively. For peripheral blood and skin, results are expressed as median and means respectively. Mann-Whitney test is used for comparisons. Results In SSc patients, circulating ILC were significantly decreased both in frequency (0.0094% of total cells [0.0005–0.052] in HD vs. 0.0024% [0.0002–0.029] in SSc, p=0.006) and in absolute counts (0.146 [0.0089–1.641] in HD vs. 0.032 [0.0049–0.427] in SSc, p=0.0005). This global decrease was mainly due to the decrease of the ILC2 frequency (0.0077% of total cells [0.0009–0.031] in HD vs. 0.0024% [0.00–0.01] in SSc, p=0.0009) and absolute counts (0.123 [0.0153–0.574] in HD vs. 0.033% [0.00–0.167] in SSc, p<0.0001). The number of ILC2 in peripheral blood was inversely correlated to skin fibrosis as assessed by the modified Rodnan skin score (r=-0.6, p=0.0041, using a Pearson test). Analysis of ILC content in total cells showed that ILC2 are present in both normal and SSc skin. However, we showed a skewing towards ILC2 (83.6% of total ILC in SSc vs. 66.4% in HD) at the expense of ILC1 (7.5% in SSc vs. 18.85% in HD) in SSc skin, while ILC3 were not affected. Conclusions SSc patients are characterized by a decrease of the ILC2 subpopulation in peripheral blood inversely correlated to skin fibrosis and a skewing of the type 1/type 2 ILC balance in the skin towards ILC2. Altogether these results show for the first time flow cytometry analysis of ILC content in whole blood and skin of SSc patients, which pave the road for further extensive phenotypic analysis. References Fichtner-Feigl S, et al. Nat Med 2006;12:99–106. Hazenberg MD, Spits H. Blood 2014;124:700–709. Truchetet ME, et al. In revision in Arthritis Rheum Disclosure of Interest None declared

FRI0378 Potent, Broad, and Specific Neutralizing Capacities of Polyclonal Anti-Interferon Alpha Antibodies Induced by IFN Kinoid in SLE Patients

Background Neovacs developed the Interferon alpha Kinoid (IFN-K), a therapeutic vaccine composed of IFNα2b coupled to a carrier protein, the keyhole limpet hemocyanin. A placebo-controlled phase I/II study was conducted comparing 3 or 4 injections of 30, 60, 120, 240 μg IFN-K versus placebo in 28 Systemic Lupus Erythematosus (SLE) patients. Anti-IFNα2b binding and neutralizing antibodies were analyzed demonstrating the immunogenicity of IFN-K towards IFNα2b. We further characterized the isotypes, avidity and specificity of anti-IFNα antibodies towards other IFN subtypes. We also compared the neutralizing capacities in sera from two SLE patient to that of 9F3 a murine anti-IFNα monoclonal antibody (Genentech patent # US 7 087 726 B2). Methods Isotype profile and cross-reactions of anti-IFNα antibodies towards IFNβ or IFNγ were tested by ELISAs. The relative anti-IFNα antibody avidity (RAV) was analyzed by ELISA in the presence of a gradient of sodium isothiocyanate chaotropic agent. Neutralizing capacities of anti-IFN antibodies towards the 13 IFNα subtypes were assessed by the compendial Madin Darby bovine kidney-vesicular stomatitis virus infection assay and were also compared to 9F3 (ATCC#PTA-2917). Results The main anti-IFNα antibody isotypes produced were IgG1 and IgG3. Anti-IFNα IgA were detected in 4 patients. Some anti-huIFNα IgM were detected mainly at day 38. IgG4 and IgE were never detected. The RAV of anti-IFNα antibodies increased with the appearance of IFNα neutralizing capacity, whereas the RAV of binding anti-IFNα antibodies without neutralizing activity remained low and stable, similar to anti-IFNα auto-antibodies detected before dosing or in the placebo group. Anti-IFNα antibodies induced by IFN-K did not cross-react with IFNβ or IFNγ. The induced anti-IFNα antibodies neutralized most or all IFNα subtypes and the number of subtypes neutralized increased with the level and persistence of neutralizing capacities. The 9F3 mab neutralized 7 IFNα subtypes, of which only two (IFNα2A and IFNα2B) strongly. In contrast, sera from the two patients exhibiting neutralizing capacities 2 years after immunizations, neutralized 13 and 9 subtypes and the neutralizing capacity of the induced polyclonal antibodies is exercised at high serum dilutions and is much more powerful against all IFNα subtypes than monoclonal 9F3 which only strongly neutralized two IFNα sub-types. Conclusions Anti-IFNα antibodies induced by IFN-K in SLE patients neutralized most or all IFNα subtypes but recognized neither IFNβ nor IFNγ. As opposed to monoclonal antibodies kinoid induced polyclonal antibodies neutralized most IFNα subtypes at high dilution. These preliminary results support the superiority of a polyclonal antibody strategy in a disease such as lupus wherein all IFNα subtypes may play a role. Disclosure of Interest G. Grouard-Vogel Shareholder of: Neovacs, Employee of: Neovacs, B. Lauwerys Consultant for: Neovacs, P. Vandepapeliere Shareholder of: Neovacs, Employee of: Neovacs, F. Colaone Shareholder of: Neovacs, Employee of: Neovacs, P. Blanco Consultant for: Neovacs, T. Defrance Consultant for: Neovacs, C. Roucairol Shareholder of: Neovacs, Employee of: Neovacs, F. Houssiau Consultant for: Neovacs DOI 10.1136/annrheumdis-2014-eular.3726