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ObjectivesTo explore the relevance of T-follicular-helper (Tfh) and pathogenic peripheral-helper T-cells (Tph) in promoting ectopic lymphoid structures (ELS) and B-cell mucosa-associated lymphoid tissue (MALT) lymphomas (MALT-L) in Sjögren’s syndrome (SS) patients.MethodsSalivary gland (SG) biopsies with matched peripheral blood were collected from four centres across the European Union. Transcriptomic (microarray and quantitative PCR) analysis, FACS T-cell immunophenotyping with intracellular cytokine detection, multicolor immune-fluorescence microscopy and in situ hybridisation were performed to characterise lesional and circulating Tfh and Tph-cells. SG-organ cultures were used to investigate functionally the blockade of T-cell costimulatory pathways on key proinflammatory cytokine production.ResultsTranscriptomic analysis in SG identified Tfh-signature, interleukin-21 (IL-21) and the inducible T-cell co-stimulator (ICOS) costimulatory pathway as the most upregulated genes in ELS+SS patients, with parotid MALT-L displaying a 400-folds increase in IL-21 mRNA. Peripheral CD4+CXC-motif chemokine receptor 5 (CXCR5)+programmed cell death protein 1 (PD1)+ICOS+ Tfh-like cells were significantly expanded in ELS+SS patients, were the main producers of IL-21, and closely correlated with circulating IgG and reduced complement C4. In the SG, lesional CD4+CD45RO+ICOS+PD1+ cells selectively infiltrated ELS+ tissues and were aberrantly expanded in parotid MALT-L. In ELS+SG and MALT-L parotids, conventional CXCR5+CD4+PD1+ICOS+Foxp3- Tfh-cells and a uniquely expanded population of CXCR5-CD4+PD1hiICOS+Foxp3- Tph-cells displayed frequent IL-21/interferon-γ double-production but poor IL-17 expression. Finally, ICOS blockade in ex vivo SG-organ cultures significantly reduced the production of IL-21 and inflammatory cytokines IL-6, IL-8 and tumour necrosis factor-α (TNF-α).ConclusionsOverall, these findings highlight Tfh and Tph-cells, IL-21 and the ICOS costimulatory pathway as key pathogenic players in SS immunopathology and exploitable therapeutic targets in SS.

Background: SDF-1/CXCL12 is a chemokine with pleiotropic functions in hematopoietic stem cell niche homeostasis, germinal center architecture, B cell maturation, neoangiogenesis, and fibrosis. Recently, the CXCL12/CXCR4/CXCR7 axis was associated with cancer metastasis and autoimmune diseases. The IgG4-related disease (IgG4-RD) is a pathological condition characterized by IgG4+ plasma cells infiltrating fibrotic lesions. The aim of this research is to investigate the relevance of SDF-1/CXCL12 in IgG4-RD.Materials and Methods: Peripheral blood samples were collected before therapy from a single-center cohort of 28 IgG4-RD patients, fulfilling the ACR-EULAR classification criteria. Clinical and serological data were obtained for each patient. In total, 14 healthy donors (NHS), 9 patients with pancreatic ductal adenocarcinoma (PDAC), and 9 with Sjogren syndrome (SSj) were recruited as controls and screened for circulating SDF-1/CXCL12 by ELISA. Moreover, paraffin-embedded pancreatic biopsies obtained from patients with IgG4-RD (n = 7), non-autoimmune pancreatitis (n = 3), PDAC (n = 5), and control tissues (n = 4) were analyzed to study the tissue expression and localization of SDF-1/CXCL12 and one of its receptors, CXCR4, and their potential relation with neutrophil extracellular traps (NETs).Results: IgG4-RD patients had higher serum levels of SDF-1/CXCL12 than normal controls (p = 0.0137). Cytokine levels did not differ between the IgG4-RD autoimmune pancreatitis (AIP) and retroperitoneal fibrosis nor between the single- and multiple-organ involvement. No correlation was seen with the IgG4-RD Responder Index, IgG4 levels, white blood cells, or inflammatory markers in the serum. When compared to SSj, the IgG4-RD AIP subgroup presents higher amounts of serum SDF-1/CXCL12 (p = 0.0275), while no differences are seen in comparison with PDAC. The expression of SDF-1/CXCL12 in the tissue was significantly higher in the IgG4-RD tissue than the normal pancreas, and the tissue with the high SDF-1/CXCL12 expression is characterized by the overall inflammatory cell infiltration, fibrosis, and high level of NETs.Conclusion: Modulating B cell development, neoangiogenesis and fibrosis, and SDF-1/CXCL12 may play a role in IgG4-RD. The higher levels observed in IgG4-RD, as compared to SSj, which closely mimics the disease, can be related to a different pattern of lesions, with prevalent fibrosis seen in IgG4-RD. Taken together, these findings suggest that drugs acting on the CXCL12/CXCR4/CXCR7 axis may affect IgG4-RD.

Chronic urticaria (CU) is a mast cell-driven disease characterized by the development of wheals, angioedema, or both for more than 6 weeks. The two major sub-types are chronic spontaneous urticaria (CSU) and inducible urticaria. In the last decade different pathophysiological mechanisms, potentially responsible for the development of the disease, have been described. It is likely that the activation of mast cells and basophils in CSU can be the results of immune system dysregulation, activation of the inflammatory cascade, and of the extrinsic coagulation pathway. Some of the mediators involved in the pathophysiological mechanisms of CSU have recently been identified as potential biomarkers useful for the diagnosis, follow-up, and management of the disease, even if they are not yet available in clinical practice. Thus, in this review we discuss new insights in the mediators involved in the pathogenesis of CSU, highlighting their potential role as biomarkers in the activity and progression of the disease and response to therapies.

Pyoderma gangrenosum (PG) is a neutrophilic dermatose (ND) characterized by a dense neutrophilic infiltrate in the affected tissue. Neutrophil extracellular traps (NETs) are web‐like structures released by neutrophils and composed of cytosolic and granule proteins assembled on a scaffold of decondensed chromatin. Very little is known about the role of NETosis in PG. Here, we assessed the possible implication of NETosis in the pathogenesis of PG by investigating the NETosis in the ulcers of 26 PG patients. We demonstrated that neutrophils in the PG skin lesions undergo an aberrant level of NETosis in 100% of the analysed cases (N = 26). All control and abscess biopsies were instead negative for the NETosis. In addition, neutrophils from peripheral blood of PG patients showed a significantly higher rate of spontaneous, but not induced, NETosis. Overall, this study suggests that the NETosis may contribute to systemic inflammation and tissue destruction in PG, thus representing a possible novel therapeutic target.

IgG4-Related Disease (IgG4-RD) is a fibroinflammatory condition characterized by a typical histopathological pattern (dense lymphoplasmacytic infiltrate with prevalent IgG4+ plasma cells and storiform fibrosis), which may involve the kidney both directly (IgG4-related kidney disease, IgG4-RKD) or indirectly, as a consequence of post-renal ureteral obstruction due to retroperitoneal fibrosis (IgG4-RD RF). The most frequent presentation of IgG4-RKD is IgG4-related tubulointerstitial nephritis (TIN), but a glomerular disease can be present, in most of the cases a membranous nephropathy. Albeit steroid-responsive, in some cases renal manifestations may lead to progressive and permanent organ damage. In this review we describe four clinical cases representative of typical and less typical renal manifestations of IgG4-RD, emphasizing a potential, subclinical, early involvement of the kidney in the disease.

Salivary glands (SGs) represent a permissive site for several sialotropic viruses whose persistence is linked to the development of autoimmunity. Natural Killer (NK) cells play a key role in viral clearance but their involvement in viral infection control and in tertiary lymphoid structures (TLS) development within SGs is unknown. By using an inducible model of TLS in the SGs of wild-type C57BL/6 mice, induced by the local delivery of a replication-defective adenovirus (AdV), we demonstrated that circulating NK cells are rapidly recruited to SGs and highly enrich the early inflammatory infiltrate prior to TLS development. NK cells migrating to SGs in response to AdV infection up-regulate NKp46, undergo proliferation, acquire cytotoxic potential, produce Granzyme-B and IFN-γ, and reduce viral load in the acute phase of the infection. Nonetheless, the selective depletion of both circulating and infiltrating NK cells in AdV-infected mice neither affect the development and frequency of TLS nor the onset of autoimmunity. These data demonstrate that, upon local viral delivery of AdV, peripheral NK cells homing to SGs can exert an early control of the viral infection but are dispensable for the formation of TLS and breach of immunologic tolerance.

Neutrophil extracellular traps (NET) formation is part of the neutrophil response to infections, but excessive or inappropriate NETosis may trigger the production of autoantibodies and cause organ damage in autoimmune disorders. Spontaneously netting neutrophils are not frequent and induction of NET in vitro by selected stimuli is necessary to investigate their structure. In the present work, the protein composition and post-translational modifications of NET produced under different stimuli have been studied by means of proteomic analysis. Neutrophils from healthy donors were stimulated by PMA, A23187, Escherichia coli LPS or untreated; after three hours, cells were washed, treated with DNase and supernatants collected for mass spectrometry. Data were analyzed by unsupervised hierarchical clustering analyses. We identified proteins contained in NETs of any source or exclusive of one stimulus: LPS-induced and spontaneous NET diverge in protein composition, while PMA- and A23187-induced NET appear more similar. Among the post-translational modifications we examined, methionine sulfoxidation is frequent especially in PMA- and LPS-induced NETs. Myeloperoxidase is the protein more extensively modified. Thus, proteomic analysis indicates that NETs induced by different stimuli are heterogeneous in terms of both protein composition and post-translational modifications, suggesting that NET induced in different conditions may have different biological effects.

Background B/T cell aggregates in the salivary glands (SG) of Sjogren9s syndrome (SS) can give rise to ectopic lymphoid structures (ELS) forming ectopic germinal centers (GCs), which has been linked to the development of MALT lymphoma (MALT-L). T follicular-helper cells (Tfh) and T follicular-regulatory cells (Tfr) are specialized CD4+ T-cells that positively and negatively regulate, respectively, the magnitude of the GCs response and the onset of autoimmunity. Objectives To characterize the infiltration of Tfh and Tfr in the SG infiltrates of patients with SS in the context of the presence/absence of ectopic GCs and in subjects with MALT-L. Methods SG biopsies with matching histology and RNA from 37 SS and 38 non-specific chronic sialadenitis (NSCS) patients were stratified as ELS-/ELS+ based on CD3/CD20/CD21/CD138 immunostaining (IHC). Histological samples and mRNA from 12 parotid MALT-L were also studied. Gene expression was measured by Taqman rt-PCR. Multicolor immunofluorescence/confocal microscopy for CD3, CD4, CD45RO, ICOS, PD1, BCL6 and FoxP3 was used to identify Tfh and Tfr. Results Tfh (CD4 + CD45RO + PD1 + ICOS + FoxP3 - ) cells and Tfr (CD4 + CD45RO + PD1 + ICOS + FoxP3 + ) cells were strongly enriched in ELS+ vs ELS- SS samples. The Tfh:Tfr ratio in ELS+ SG was approximately 2:1. Interestingly, while in tonsils Tfr were routinely detected within GCs, in ELS+ SG Tfr were predominantly excluded from the B cell follicles and accumulated in the T cell rich areas at the periphery of the lymphoid aggregates. Conversely, Tfh densely infiltrated the B cell rich areas and, within ectopic GCs, acquired BCL6. Furthermore, Tfh infiltration closely correlated with SG IL-21 mRNA expression, which in turn was strongly correlated with CD3, CD20 and CD138 IHC scores and with CXCL13, LTb, BAFF, AID and Pax5 gene expression. Finally, MALT-L samples displayed 10-fold higher IL-21 mRNA and twice as much PD1 + ICOS + BCL6 + Tfh-cells/field compared to ELS+ SS samples. Conclusions Within the SG of SS patients Tfh cells closely segregate with lesional IL-21 expression, localize within ELS and are strongly enriched during MALT-L development. Conversely, although Tfr cells are also recruited to ELS+ SG in SS patients, we consistently demonstrated follicular exclusion of this subset from ectopic GCs. This suggests that Tfr in SS SG fail to exert their physiological immunoregulatory properties in controlling the magnitude of the GCs response and B cell autoreactivity, as observed in tonsils. Acknowledgements This work was supported by project grants from the Medical Research Council (MR/N003063/1 to MB) and Arthritis Research UK (grant 20089 to MB). Disclosure of Interest E. Pontarini: None declared, W. Murray-Brown: None declared, C. Croia: None declared, E. Astorri: None declared, D. Lucchesi: None declared, N. Lepse: None declared, N. Sutcliffe: None declared, A. Tappuni: None declared, C. Pitzalis: None declared, M. Bombardieri Consultant for: Amgen/Medimmune, GSK and UCB

Objective To examine whether the B cell tropic γ-herpesvirus Epstein-Barr virus (EBV) is aberrantly expressed in its latent and lytic forms within ectopic lymphoid structures (ELS) in the salivary glands of patients with Sjogren's syndrome (SS), and to investigate the relationship between EBV dysregulation, B cell activation, in situ differentiation of autoreactive plasma cells, disease-specific autoantibody production, and cytotoxicity. Methods Latent and lytic EBV infection in the salivary glands of 28 patients with SS and 38 patients with nonspecific chronic sialadenitis (NSCS), characterized for the presence or absence of ELS, was investigated by reverse transcription–polymerase chain reaction, in situ hybridization, and immunohistochemical/immunofluorescence staining. Glandular versus synovial production of anti−Ro 52, anti–citrullinated protein antibodies (ACPAs), and anti–EBV peptide antibodies was analyzed in situ or in vivo in human SS/SCID and human rheumatoid arthritis/SCID mouse chimeras. Results EBV dysregulation within inflammatory infiltrates was observed exclusively in ELS+ SS salivary gland tissue, as revealed by latent EBV infection and lytic EBV infection in B cells and plasma cells, respectively. Conversely, epithelial latent membrane protein 2A expression was observed in both patients with SS and patients with NSCS. Importantly, perifollicular plasma cells displaying Ro 52 immunoreactivity were frequently infected by EBV. Furthermore, ELS-containing SS salivary gland tissue that was transplanted into SCID mice supported the production of anti–Ro 52/anti–La 48 and anti-EBV antibodies but not ACPAs. Analysis of CD4+ and CD8+ T cell localization and granzyme B expression demonstrated that the persistence of EBV in ELS-containing SS salivary glands was associated with follicular exclusion of CD8+ T cells and impaired CD8-mediated cytotoxicity. Conclusion Active EBV infection is selectively associated with ELS in the salivary glands of patients with SS and appears to contribute to local growth and differentiation of disease-specific autoreactive B cells.

Fibromyalgia (FM) is thought to occur because of the combination of interactions among neurotransmitters, such as neuropeptide Y (NPY), stressors, hormones, cytokines, and both the immune and sympathetic nervous systems. The aim of this study was to evaluate serum concentrations of cytokines, antipolymer antibodies (APA), and NPY in 51 patients with FM, 25 with tension-type headache (TTH), and 15 healthy controls. Serum concentrations of eight different cytokines, APA and NPY, were measured. Interleukin (IL)-1RA, IL-6, IL-10, and tumor necrosis factor-alpha were higher in serum of FM patients compared with TTH patients and a significant correlation between IL-10 and Fibromyalgia Impact Questionnaire score was observed. There was a significant difference between FM and TTH versus controls in NPY levels, but not in APA levels. Cytokines and NPY take part in pain modulation and even if they are altered in FM they cannot be considered as measurable biomarkers of disease.

During the last decade, three tumour necrosis factor (TNF)α antagonists have been approved for the treatment of moderate to severe rheumatoid arthritis (RA).1 These new drugs, while extremely powerful, can induce puzzling biological phenomena such as the production of autoantibodies whose extent seems to vary accordingly to the drug used.2,3 A dysregulation of apoptosis with exposition of nuclear antigens and consequent rapid (within hours) accumulation of nucleosomes has been evoked to explain autoantibody occurrence in patients treated with infliximab.4 In addition (or in alternative) to an increased release of nuclear antigens, a defect in clearance mechanisms can play a role in the generation of autoantibodies.5 We studied autoantibody profiles (anti-nuclear antibody (ANA), anti-double stranded …

To examine whether the B cell tropic γ-herpesvirus Epstein-Barr virus (EBV) is aberrantly expressed in its latent and lytic forms within ectopic lymphoid structures (ELS) in the salivary glands of patients with Sjögren's syndrome (SS), and to investigate the relationship between EBV dysregulation, B cell activation, in situ differentiation of autoreactive plasma cells, disease-specific autoantibody production, and cytotoxicity.Latent and lytic EBV infection in the salivary glands of 28 patients with SS and 38 patients with nonspecific chronic sialadenitis (NSCS), characterized for the presence or absence of ELS, was investigated by reverse transcription-polymerase chain reaction, in situ hybridization, and immunohistochemical/immunofluorescence staining. Glandular versus synovial production of anti-Ro 52, anti-citrullinated protein antibodies (ACPAs), and anti-EBV peptide antibodies was analyzed in situ or in vivo in human SS/SCID and human rheumatoid arthritis/SCID mouse chimeras.EBV dysregulation within inflammatory infiltrates was observed exclusively in ELS+ SS salivary gland tissue, as revealed by latent EBV infection and lytic EBV infection in B cells and plasma cells, respectively. Conversely, epithelial latent membrane protein 2A expression was observed in both patients with SS and patients with NSCS. Importantly, perifollicular plasma cells displaying Ro 52 immunoreactivity were frequently infected by EBV. Furthermore, ELS-containing SS salivary gland tissue that was transplanted into SCID mice supported the production of anti-Ro 52/anti-La 48 and anti-EBV antibodies but not ACPAs. Analysis of CD4+ and CD8+ T cell localization and granzyme B expression demonstrated that the persistence of EBV in ELS-containing SS salivary glands was associated with follicular exclusion of CD8+ T cells and impaired CD8-mediated cytotoxicity.Active EBV infection is selectively associated with ELS in the salivary glands of patients with SS and appears to contribute to local growth and differentiation of disease-specific autoreactive B cells.

Objectives Rheumatoid arthritis (RA) is associated with an increased Epstein–Barr virus (EBV) blood DNA load, a robust immune response to EBV and cross-reactive circulating antibodies to viral and self-antigens. However, the role of EBV in RA pathogenesis remains elusive. Here, we investigated the relationship between synovial EBV infection, ectopic lymphoid structures (ELS) and immunity to citrullinated self and EBV proteins. Methods Latent and lytic EBV infection was investigated in 43 RA synovial tissues characterised for presence/absence of ELS and in 11 control osteoarthritis synovia using RT-PCR, in situ hybridisation and immunohistochemistry. Synovial production of anti-citrullinated protein (ACPA) and anti-citrullinated EBV peptide (VCP1/VCP2) antibodies was investigated in situ and in vivo in the severe combined immunodeficiency (SCID)/RA chimeric model. Results EBV dysregulation was observed exclusively in ELS+ RA but not osteoarthritis (OA) synovia, as revealed by presence of EBV latent (LMP2A, EBV-encoded small RNA (EBER)) transcripts, EBER+ cells and immunoreactivity for EBV latent (LMP1, LMP2A) and lytic (BFRF1) antigens in ELS-associated B cells and plasma cells, respectively. Importantly, a large proportion of ACPA-producing plasma cells surrounding synovial germinal centres were infected with EBV. Furthermore, ELS-containing RA synovia transplanted into SCID mice supported production of ACPA and anti-VCP1/VCP2 antibodies. Analysis of CD4+ and CD8+ T-cell localisation and granzyme B expression suggests that EBV persistence in ELS-containing synovia may be favoured by exclusion of CD8+ T cells from B-cell follicles and impaired CD8-mediated cytotoxicity. Conclusions We demonstrated active EBV infection within ELS in the RA synovium in association with local differentiation of ACPA-reactive B cells.

SummaryTo evaluate the anti-endothelial cell antibodies (AECA), anti-cardiolipin antibodies (aCL) and serum mannose-binding lectin (MBL) profiles of a large cohort of Yemeni patients with rheumatic heart disease (RHD) and to correlate these findings with clinical features of the disease. Patients (n = 140) were recruited from Al-Thawra Hospital in Sana'a, Yemen. All had RHD diagnosed according to modified Jones' criteria. We also studied 140 sex- and age-matched healthy blood donors from the same area. Echocardiography was performed according to the recommendations of the American Society of Echocardiography. Solid phase enzyme-linked immunosorbent assays (ELISAs) were used to measure AECA and aCL titres and serum MBL levels. Forty per cent of the patients were AECA-positive, but only 7·8% were positive for aCL antibodies. Serum MBL levels were significantly lower in the RHD group (median 4221 ng/ml versus 5166 ng/ml in healthy controls). AECA titres were correlated positively with patient age, duration of RHD and the severity of aortic stenosis, as determined by echocardiographic findings. In several autoimmune rheumatic diseases, such as systemic lupus erythematosus, vasculitis and scleroderma, AECA have been shown to play pathogenic roles by producing proinflammatory and procoagulant effects (increased expression of adhesion molecules and tissue factors, increased cytokine release) in endothelial cells. In RHD, these autoantibodies might represent a pathological link between activation of the valvular endothelium and valvular damage.

Background Immunoglobulin G4 (IgG4)-related disease (IgG4-RD) is an immune-mediated condition characterized by dense IgG4+ plasmacytic infiltration of diverse organs, fibrosis, tumefactive lesions and eosinophilia. Salivary and lacrimal glands are frequent targets that can present with enlargement and severe hypofunction. The diagnosis of IgG4-RD is challenging because of its serological and histopathological overlaps with other diseases, in particular, a differential diagnosis with Sjogren9s syndrome (SS) can sometimes be achieved only on the basis of a labial salivary gland biopsy. Objectives In this study we evaluated whether some of the patients with sicca symptoms not fulfilling SS classification criteria, were in fact affected by IgG4-RD. Methods A total of 81 Sicca and 50 SS patients were retrospectively evaluated for IgG4-RD-associated signs and symptoms. Additionally, IgG4 and total-IgG staining was performed on lip gland biopsies from all patients while IgG4 serum levels were evaluated on the Italian cohort. Results Only 2 of the Sicca patients had positive IgG4 staining but did not fulfil the diagnostic criteria. The serum analysis found 3 sicca patients with raised IgG4 (≥1.35 g/L) but none had a concomitant positive histology nor IgG4-RD related symptoms. Conclusions In our cohort of patients with sicca symptoms not fulfilling SS criteria no case could be accounted as an undiagnosed IgG4-RD based upon IgG4 staining on labial salivary gland biopsies. This study also suggests that in the absence of a clear clinical suspicious of an IgG4-RD, a routine IgG4 staining on labial salivary gland biopsies is not of clinical utility. References Yamamoto M, Harada S, Ohara M, Suzuki C, Naishiro Y, Yamamoto H, et al. Clinical and pathological differences between Mikulicz9s disease and Sjogren9s syndrome. Rheumatology (Oxford). 2005 Feb;44(2):227-34. PubMed PMID: 15509627. Umehara H, Okazaki K, Masaki Y, Kawano M, Yamamoto M, Saeki T, et al. Comprehensive diagnostic criteria for IgG4-related disease (IgG4-RD), 2011. Mod Rheumatol. 2012 Feb;22(1):21-30. PubMed PMID: 22218969. Disclosure of Interest None declared

Background The ubiquitous □-herpesvirus Epstein-Barr virus (EBV) infects B cells and modifies their differentiation programme leading to B cell activation and immortalization. Due to these unique properties, EBV has been suggested to play a role in rescuing autoreactive B cells and promoting autoimmunity. Although different evidences support a link between EBV infection and Sjogren’s syndrome (SS), the exact role of EBV in SS pathogenesis remains elusive. Objectives To investigate the potential role of EBV in SS pathogenesis by analysing EBV infection in the SS salivary glands and its relationship with ectopic lymphoid structures (ELS), in situ autoreactive plasma cell differentiation, autoantibody production and cytotoxic immune response in the salivary glands. Methods Latent and lytic EBV infection was investigated in SS and non-specific chronic sialoadenitis (NSCS) labial salivary glands characterised for presence/absence of ELS by RT-PCR, in situ hybridization and immunohistochemistry/immunofluorescence. Glandular production of anti-Ro52 antibodies and anti-EBV peptides antibodies was investigated in situ and/or in vivo in a novel SCID/SS chimeric model. Results EBV dysregulation was observed exclusively in ELS+ SS salivary glands as revealed by presence EBV-encoded small RNA (EBER) transcripts and EBER+ cells and immunoreactivity for EBV latent (LMP1, LMP2A) and lytic (BFRF1) antigens in ELS-associated B cells and plasma cells, respectively. Importantly, a subset of EBV+ peri-follicular plasma cells produced anti-Ro52 antibodies. Furthermore, ELS-containing SS salivary glands transplanted into SCID mice supported the production of anti-Ro52, anti-La and anti-EBV antibodies. Analysis of CD4+ and CD8+ T-cell localization and granzyme B expression suggested that EBV persistence in ELS-containing SS salivary glands is favoured by exclusion of CD8+ T cells from B-cell follicles and impaired CD8-mediated cytotoxicity. Conclusions We demonstrated latent and active EBV infection within ELS in the SS salivary glands that appears to contribute to local growth and differentiation of autoreactive B cells producing SS-associated autoantibodies. Disclosure of Interest : None Declared

Background Ectopic lymphoid structures (ELS) are preferential sites for Epstein-Barr virus (EBV) latency and reactivation in the brain of multiple sclerosis (1) and are functional sites of autoreactive B cell activation in the synovial tissue of rheumatoid arthritis (RA) patients (2) leading to the in situ production of anti-citrullinated protein/peptide antibodies (ACPA). However, it is unknown whether ELS are responsible for the generation of antibodies against native and/or post-translationally modified EBV proteins. Objectives In this work we aimed: 1) To investigate the presence of anti-VCA, anti-EBNA1, anti-EA and ACPA antibodies in the serum vs synovial fluid (SF) of RA patients; 2) To provide in vivo evidence of the synovial production of antibodies against unmodified and citrullinated EBV-proteins (3) in the HuRA/SCID chimeric model; 3) To investigate whether EBV-infected synovial plasma cells produce ACPA. Methods Anti-EBNA1, anti-VCA, anti-EA and ACPA IgG were measured in paired serum and SF from RA and OA patients. The local production of unmodified and citrullinated (VCP1 and VCP2) anti-EBV antibodies (3) was investigated by screening the serum of 30 SCID mice transplanted with ELS+ vs ELS- RA synovium. Finally, sequential double immunofluorescence for biotinylated citrullinated fibrinogen (BCf), BFRF1 (EBV lytic antigen) and CD138 was used to detect EBV reactivation in ACPA-producing synovial plasma cells. Results Serum and SF levels of anti-EBNA1, anti-VCA and ACPA IgG were significantly correlated. Sera of SCID mice transplanted with ELS+ (but not ELS-) RA synovia displayed high levels of human IgG ACPA, anti-EBNA1 and anti-VCA but not anti-EA antibodies. Interestingly, a proportion of SCID mice sera displaying high reactivity against anti-CCP and anti-EBNA1 antibodies were also found positive for anti-VCP1 and VCP2. Finally, in ELS+ RA synovia a subset of locally differentiated ACPA+ plasma cells surrounding ectopic germinal centres displayed EBV lytic infection as detected by reactivity for BFRF1. Conclusions Here we showed that antibodies against unmodified and citrullinated EBV antigens are locally produced in the RA synovium in the presence of ELS and are closely associated with in situ ACPA production. Evidence of ACPA+ plasma cells showing lytic EBV infection strongly supports an important role for EBV in the activation and differentiation of autoreactive B cells within the RA synovium. References Serafini B, Rosicarelli B, Franciotta D, Magliozzi R, Reynolds R, Cinque P, Andreoni L, Trivedi P, Salvetti M, Faggioni A, Aloisi F. Dysregulated Epstein-Barr virus infection in the multiple sclerosis brain. J Exp Med. 2007 Nov 26;204(12):2899-912. Humby F, Bombardieri M, Manzo A, Kelly S, Blades MC, Kirkham B, Spencer J, Pitzalis C. Ectopic lymphoid structures support ongoing production of class-switched autoantibodies in rheumatoid synovium. PLoS Med. 2009 Jan 13;6(1):e1 Pratesi F, Tommasi C, Anzilotti C, Chimenti D, Migliorini P. Deiminated Epstein-Barr virus nuclear antigen 1 is a target of anti-citrullinated protein antibodies in rheumatoid arthritis. Arthritis Rheum. 2006 Mar;54(3):733-41. Disclosure of Interest None Declared

Background IL-21 is a pro-inflammatory cytokine that plays a key role in the activation and differentiation of B cells 1 . B cells infiltrate the salivary glands (SG) of Sjogren’s syndrome (SS), an autoimmune disease characterized by immune infiltration in the lacrimal and salivary glands leading to exocrine dysfunction 2 . In SS SG, B cells are organized as functional ectopic germinal centers (GC) and are pivotal in SS pathogenesis 3 . Follicular helper T cells (Tfh) abundantly express IL-21 and contribute to GC B cell affinity maturation via induction of AID, somatic hypermutation and class switching 4 . Objectives We aimed to investigate IL-21 mRNA expression in SS SG, to correlate expression with markers of inflammation such as CXCL13, Ltb, BAFF as well as markers of B cell differentiation AID, Pax5 and Blimp1, and then to assess the relationship of IL-21 and Tfh cells with the development of functional ectopic GC. Methods IL-21 and IL-21R mRNA expression was assessed by Taqman PCR in 22 labial SG of patients with SS and 17 with non-specific chronic sialadenitis (NSCS). Expression levels were correlated with genes regulating ectopic GC formation such as CXCL13 and Ltb and B cell function such as BAFF, AID, Pax5 and Blimp1. In addition, GC formation in the SG was assessed by IHC for B/T cell segregation and follicular dendritic cell (FDC) networks. Finally, Tfh cells infiltration in the SG was assessed by IF for CD3/CD45RO/ICOS and PD1. Results SG of SS patients displayed higher expression of IL-21 and IL-21R mRNA compared to NSCS (mean fold increase±SEM 38±12 vs 5±4, p=0.02 for IL-21 and 59±13 vs 12±2, p=0.01). In SS, IL-21 mRNA strictly correlated with the levels of CXCL13 (Spearman’s r=0.697, p Conclusions Here we show that IL-21 expression is significantly increased in the SG of SS patients and strongly correlates with ectopic GC formation, functional B cell activation and accumulation of Tfh. These data strongly implicate IL-21 and Tfh cells in the development and maintenance of functional ectopic GC in the SG and could represent novel therapeutic targets in SS. References Mehta, D. S., et al. , (2004). Immunological Reviews Christodoulou, M. I., et al., (2010) . Journal of Autoimmunity Barone, F., et al., (2005) . Arthritis and Rheumatism Crotty, S. (2011) . Annual Review of Immunology Disclosure of Interest None Declared

Objectives Rheumatoid arthritis (RA) is associated with an increased Epstein-Barr virus (EBV) blood DNA load, a robust immune response to EBV and cross-reactive circulating antibodies for viral and self-antigens. However, the role of EBV in RA pathogenesis remains elusive. Here we investigated the relationship between synovial EBV infection, ectopic lymphoid structures (ELS) and immunity to citrullinated self and EBV proteins. Methods Latent and lytic EBV infection was investigated in 43 RA synovial tissues characterised for presence/absence of ELS and 11 OA samples by RT-PCR, in situ hybridisation and immunohistochemistry/immunofluorescence. Synovial production of anti-citrullinated proteins (ACPA) and anti-citrullinated EBV peptides (VCP1/VCP2) antibodies was investigated in situ or in vivo in the SCID/RA chimeric model. Results EBV dysregulation was observed exclusively in ELS+ RA, but not OA, synovia as revealed by presence of EBV latent [LMP2A, EBV-encoded small RNA (EBER)] transcripts and EBER+ cells and immunoreactivity for EBV latent (LMP1, LMP2A) and lytic (BFRF1) antigens in ELS-associated B cells and plasma cells, respectively. Importantly, ~20% of synovial plasma cells producing ACPA were infected with EBV. Furthermore, ELS-containing RA synovia transplanted into SCID mice supported production of ACPA and anti-VCP1/VCP2 antibodies cross-recognised by ACPA. Analysis of CD4+ and CD8+ T-cell localisation and granzyme B expression suggests that EBV persistence in ELS-containing synovia is favoured by exclusion of CD8+ T cells from B-cell follicles and impaired CD8-mediated cytotoxicity. Conclusions We demonstrated active EBV infection within ELS in the RA synovium that appears to contribute to local growth and differentiation of ACPA-reactive B cells.