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Summary Background Primary Sjogren's syndrome is a systemic autoimmune disease characterised by secretory gland dysfunction, for which no effective therapy is available. Based on the complementary properties of leflunomide and hydroxychloroquine in inhibiting activation of key immune cells in primary Sjogren's syndrome, we aimed to evaluate the clinical efficacy and safety of leflunomide–hydroxychloroquine combination therapy in patients with primary Sjogren's syndrome. Methods We did a placebo-controlled, double-blinded, phase 2A randomised clinical trial in patients with primary Sjogren's syndrome at the University Medical Center Utrecht (Utrecht, Netherlands). Eligible patients were aged 18–75 years, had a European League Against Rheumatism (EULAR) Sjogren's syndrome disease activity index (ESSDAI) score of 5 or higher, and a lymphocytic focus score of 1 or higher in labial salivary gland biopsy specimens. Patients were randomly assigned (2:1) with block randomisation (block size of six) to receive leflunomide 20 mg and hydroxychloroquine 400 mg daily or placebo for 24 weeks. The primary endpoint was the between-group difference in change in ESSDAI scores from 0 to 24 weeks, adjusted for baseline ESSDAI score. Patients were analysed according to the intention-to-treat principle. This study is registered with EudraCT, 2014–003140–12. Findings Between March 7, 2016, and Nov 30, 2017, 37 patients were screened, of whom 29 patients (28 women and one man) were enrolled. 21 patients were assigned to receive leflunomide–hydroxychloroquine and eight patients were assigned to receive placebo. One patient in the placebo group required high-dose prednisone to treat polymyalgia rheumatica at week 13 and was excluded from the primary analysis. From 0 to 24 weeks, the mean difference in ESSDAI score, adjusted for baseline values, in the leflunomide–hydroxychloroquine group compared with the placebo group was −4·35 points (95% CI −7·45 to −1·25, p=0·0078). No serious adverse events occurred in the leflunomide–hydroxychloroquine group and two serious adverse events occurred in the placebo group (hospital admission for pancreatitis and hospital admission for nephrolithiasis). The most common adverse events in the leflunomide–hydroxychloroquine group were gastrointestinal discomfort (11 patients [52%] vs two [25%] in the placebo group), modest transient increases in alanine aminotransferase (ten [48%] vs one [13%]), and short episodes of general malaise and shivering (nine [43%] vs one [13%]). Interpretation Leflunomide–hydroxychloroquine was safe and resulted in a clinical response in patients with primary Sjogren's syndrome. These results warrant further evaluation of leflunomide–hydroxychloroquine combination therapy in larger clinical trials. Funding ZonMw.

Background Sjögren’s syndrome is a systemic autoimmune disease characterized by immune cells predominantly infiltrating the exocrine glands and frequently forming ectopic lymphoid structures. These structures drive a local functional immune response culminating in autoantibody production and tissue damage, associated with severe dryness of mucosal surfaces and salivary gland hypofunction. Cenerimod, a potent, selective and orally active sphingosine-1-phosphate receptor 1 modulator, inhibits the egress of lymphocytes into the circulation. Based on the mechanism of action of cenerimod, its efficacy was evaluated in two mouse models of Sjögren’s syndrome. Methods Cenerimod was administered in two established models of Sjögren’s syndrome; firstly, in an inducible acute viral sialadenitis model in C57BL/6 mice, and, secondly, in the spontaneous chronic sialadenitis MRL/lpr mouse model. The effects of cenerimod treatment were then evaluated by flow cytometry, immunohistochemistry, histopathology and immunoassays. Comparisons between groups were made using a Mann-Whitney test. Results In the viral sialadenitis model, cenerimod treatment reduced salivary gland immune infiltrates, leading to the disaggregation of ectopic lymphoid structures, reduced salivary gland inflammation and preserved organ function. In the MRL/lpr mouse model, cenerimod treatment decreased salivary gland inflammation and reduced T cells and proliferating plasma cells within salivary gland ectopic lymphoid structures, resulting in diminished disease-relevant autoantibodies within the salivary glands. Conclusions Taken together, these results suggest that cenerimod can reduce the overall autoimmune response and improve clinical parameters in the salivary glands in models of Sjögren’s syndrome and consequently may reduce histological and clinical parameters associated with the disease in patients.

Significance TLS, which are clusters of lymphocytes and stromal cells observed at sites of chronic inflammation, play a key role in sustaining disease progression in autoimmune conditions. While the role of lymphocytes in these structures has been studied extensively, the role of fibroblasts, nonhematopoietic stromal cells, in the formation and maintenance of TLS has not been demonstrated. Here, we establish that, at sites of TLS establishment, resident fibroblasts expand and acquire immunological features in a process that is dependent on IL13 and IL22. Interference with this process or depletion of immunofibroblasts leads to involution of TLS, resulting in decreased immune-cell activation and resolution of tissue pathology, thus supporting the use of fibroblast-targeting strategies to treat TLS-associated autoimmune diseases., Resident fibroblasts at sites of infection, chronic inflammation, or cancer undergo phenotypic and functional changes to support leukocyte migration and, in some cases, aggregation into tertiary lymphoid structures (TLS). The molecular programming that shapes these changes and the functional requirements of this population in TLS development are unclear. Here, we demonstrate that external triggers at mucosal sites are able to induce the progressive differentiation of a population of podoplanin (pdpn)-positive stromal cells into a network of immunofibroblasts that are able to support the earliest phases of TLS establishment. This program of events, that precedes lymphocyte infiltration in the tissue, is mediated by paracrine and autocrine signals mainly regulated by IL13. This initial fibroblast network is expanded and stabilized, once lymphocytes are recruited, by the local production of the cytokines IL22 and lymphotoxin. Interfering with this regulated program of events or depleting the immunofibroblasts in vivo results in abrogation of local pathology, demonstrating the functional role of immunofibroblasts in supporting TLS maintenance in the tissue and suggesting novel therapeutic targets in TLS-associated diseases.

Backgrounds The organization of minor salivary glands (MSG) infiltrates, in patients with Sjögren’s syndrome (SS), associates with disease severity and progression. Aberrant regulation of lymphocyte autophagy is involved in autoimmunity, and in previous work, we provided the first evidence of upregulated autophagy in CD4+ T cells infiltrating SS MSG. The aim of this study was to further explore autophagy in SS infiltrating and circulating lymphocytes and to investigate its role in disease histopathological progression. Methods After collection of 20 SS MSG, the presence of lymphocyte aggregates (foci) and the formation of germinal center (GC)-like structures were observed by H&E and confirmed by immunohistochemistry. The expression of autophagy-related genes, Atg5 and MAP1LC3A, was detected by RT-PCR on microdissected salivary gland tissue and control tonsils. In MSG and tonsils, autophagic lymphocytes were identified by the detection of the autophagosome protein LC3B visualized as LC3 puncta staining by immunofluorescence. Peripheral blood autophagy was assessed by flow cytometry in SS and healthy controls (HC). Results Real-time PCR demonstrated higher expression in the autophagy genes Atg5 and MAP1LC3A in MSG GCs as compared to both small foci (p = 0.0075, p = 0.0002) and GCs from tonsils (p = 0.0001, p = 0.0037). In MSG, LC3 puncta staining was detectable on both CD3+ and CD20+ lymphocytes; in tonsils, LC3 puncta was almost undetectable on all lymphocytes. Compared to HC (n = 20), flow cytometry did not reveal any increase of autophagy in SS circulating lymphocytes (n = 30). Conclusions In SS MSG, lymphocytes’ autophagy is a feature of infiltrating T and B cells and is associated with histological severity. Interestingly, in MSG aberrant regulation of autophagy is detectable in GC-like structures possibly indicating its involvement in the development and persistence of the autoimmune process within the lesions.

Background: Autophagy is a lysosome mediated catabolic process that promotes cell survival during stress conditions. Aberrant regulation of autophagy is involved in the pathogenesis of a widening spectrum of autoimmune disorders: in particular, activation of autophagy pathway is implicated in aberrant survival of activated T cells. We recently demonstrated that autophagy is up-regulated in CD4+ T lymphocytes from patients with Sjogren’s Syndrome (SS) both in peripheral blood and tissue. Moreover, aberrant autophagy correlates with systemic disease activity. Little is known on the role of autophagy in B cells homeostasis in SS. Objectives: To investigate the activation of the autophagy pathway in lymphocytes infiltrating salivary gland in SS and peripheral blood from patients with SS. Methods: Frozen SS minor salivary glands (MSG) from 20 patients were sectioned and evaluated with immunohistochemistry (IHC) by staining for CD3+, CD20+, and CD21+. MSG sections, sequential to IHC, were stained by Cresyl Violet and microdissected (Laser Capture Microdissection) in order to isolate and collect small infiltrates, large CD21- infiltrates, and large CD21+ infiltrates (GC-like structures). Microdissected GC from frozen human tonsils were used as control. The expression of autophagy genes Atg5 and MAP1LC3II (expressed as 2^deltaCT normalized to GADPH) was determined by qPCR on microdissected tissue. For studies on peripheral blood, 19 SS patients and 11 healthy controls were enrolled, and autophagy, expressed as the ratio between Mean Fluorescence Intensity and the isotopic control (rMFI), was evaluated by Cyto-ID® Autophagy detection kit in T lymphocytes (CD4+ and CD8+) and B lymphocytes (CD19+) using a FACSCalibur cytometer. Results: Expression of the autophagy genes Atg5 and MAP1LC3 was significantly higher in the GCs from SS salivary glands compared to control tonsils (p Immunophenotyping studies revealed activation of the autophagy pathway in both circulating CD19+ and CD4+ lymphocytes from SS (p=0.04 and p=0.03 compared to HC); conversely, no pathway activation was detected in CD8+ cells. Of note, in patients with SS activation of autophagy was most marked in circulating CD19+ B cells compared the other subsets [(CD19+ vs CD4+ (p Conclusion: Our findings revealed that the autophagy pathway is aberrantly activated in lymphocytes infiltrating salivary glands of SS patients. Of note, progressively higher expression levels of autophagy genes mirrored the severity and extension of inflammatory infiltrates. Immunophenotyping studies revealed that activation of autophagy is preeminent in B cells. Thus, activation of autophagy emerges as a feature of both infiltrating and circulating SS lymphocytes, and as mechanism possibly implicated in the activation and survival o?f autoreactive cells. Disclosure of Interests: Serena Colafrancesco: None declared, Antonina Minniti: None declared, Roberta Priori: None declared, Marta Vomero: None declared, cristiana barbati: None declared, Francesca Arienzo: None declared, Valentina Iannizzotto: None declared, Elena Pipi: None declared, Joana Campos: None declared, Saba Nayar: None declared, Francesco Ciccia Grant/research support from: CELGENE, PFIZER, Consultant for: UCB, NOVARTIS, CELGENE, PFIZER, LILLY, Paid instructor for: UCB, NOVARTIS, CELGENE, PFIZER, LILLY, JANSSEN, Speakers bureau: UCB, NOVARTIS, CELGENE, PFIZER, LILLY, JANSSEN, MSD, ROCHE, AMGEN, Francesca Barone Grant/research support from: GlaxoSmithKline, Roche, UCB Pharma, Actelion, ONO Pharmaceutical, Consultant for: GlaxoSmithKline, Roche, Actelion, ONO Pharmaceutical, Guido Valesini: None declared, cristiano alessandri: None declared

Background Fatigue is the key symptom that leads to poor health related quality of life and loss of work productivity in patients with primary Sjogren’s syndrome (pSS). RSLV-132 is a first-in-class drug comprising RNase1 fused to the Fc region of IgG1. It is designed to increase serum RNase activity to digest RNA associated immune complexes, thereby reducing Toll-like receptor (TLR) activation and subsequent production of type 1 interferon (IFN), B-cell proliferation, and autoantibody production – mechanisms that are key to pSS pathogenesis. IFN pathway dysregulation has also been implicated in fatigue. Objectives To explore the clinical efficacy of RSLV-132 in improving patient-reported outcomes (PRO), particularly fatigue among patients with pSS. Methods PSS patients with positive anti-Ro and IFN gene expression signature were randomised 3:1 to RSLV-132 10 mg/kg IV or placebo (PBO) at weeks 0,1, 2, and then fortnightly until week 12. Use of hydroxycholoroquine, other immunomodulatory therapies or prednisolone > 10mg daily were not permitted. There was no minimum entry criteria for EULAR Sjogren’s syndrome disease activity (ESSDAI) or EULAR Sjogren’s syndrome patient reported index (ESSPRI). The primary endpoint was changes in gene or protein expression levels in blood indicative of reduced inflammation. Secondary endpoints, among others, included safety and tolerability, and changes in PRO between baseline and week 14 including ESSPRI, FACIT, Fatigue visual analogue score (0-100), Profile of fatigue (PRO-F (0-7)) and Neuropsychological tests. Results Thirty patients were randomised (RSLV=22, PBO=8). Baseline clinical and demographic characteristics were comparable between groups. Two subjects randomised to active drug withdrew from the study before dosing. Among the patients receiving RSLV-132, the mental component of PRO-F improved by 1.53 points compared to worsening of 0.06 points in the PBO group (p=0.046). Consistently, there was a significant improvement in the RSLV-132 group in their Digital Symbol Substitution Test performance with a reduction of 16.4s in completing the test compared to an increase of 2.8s in the PBO group (p=0.024). The physical component of PRO-F improved by 0.8 points in the RSLV-132 group compared to improvement of 0.06 points in the PBO group (p=0.142). Similar trends were observed for ESSPRI and FACIT-F scores. There was no drug effect on ESSDAI but the baseline median ESSDAI scores were low for both groups (RSLV=3 (IQR (0,11), PBO=5 (IQR (0,18)). Treatment Emergent Adverse Events were reported by all participants and similar between arms; overall RSLV-132 was safe and well-tolerated. One SAE of parotitis in the RSLV-132 arm occurred 88 days after last dose of study drug and was considered unrelated to the RSLV-132. Analyses of the primary endpoints and other secondary endpoints are ongoing. Conclusion RSLV-132 is a promising therapy to improve the symptoms of fatigue in patients with pSS, with a good safety profile. Further investigation of its use in pSS is warranted. Reference [1] Study supported by Resolve Therapeutics Disclosure of Interests Benjamin Fisher Consultant for: Novartis, Roche, MedImmune and BMS, Francesca Barone Grant/research support from: GlaxoSmithKline, Roche, UCB Pharma, Actelion, ONO Pharmaceutical, Consultant for: GlaxoSmithKline, Roche, Actelion, ONO Pharmaceutical, Kerry Jobling: None declared, Peter Gallagher: None declared, Victoria Macrae: None declared, Andrew Filby: None declared, Gillian Hulmes: None declared, Paul Milne: None declared, Emmanuella Traianos: None declared, Valentina Iannizzotto@bham.ac.uk: None declared, Alexandre Dumusc: None declared, Simon J. Bowman Grant/research support from: Previously UCB Pharma (to University of Birmingham) and Roche, Consultant for: 2016-7: Novartis, Mitsubishi Tanabe Pharma 2017-8: AstraZeneca, MedImmune, GFK, Xtlbio, ONO Pharmaceutical 2018-9: Novartis, AstraZeneca, UCB Pharma, Jessica Tarn: None declared, Dennis Lendrem: None declared, Daniel Burge Employee of: Resolve Therapeutics, Medical Monitor for the clinical trial, James Posada Shareholder of: Owns equity of Resolve Therapeutics, Employee of: Founder and CEO of Resolve Therapeutics, Wan-fai Ng Grant/research support from: I have research collaborations with Resolve Therapeutics, electroCore, GSK, and Abbvie., Consultant for: Novartis, GSK, Abbvie, MedImmune, Pfizer, BMS

Career situation of first and presenting author Post-doctoral fellow. Introduction Sjogren’s syndrome (SS) is an autoimmune condition characterised by systemic B cell activation, autoantibody production and ectopic germinal centers (GC) formation within salivary gland (SG). The extent of SG infiltrate has been proposed as biomarker of disease severity. Plasma levels of CXCL13 correlate with GC activity in animal models and disease severity in SS, suggesting its potential use as a surrogate serum marker to monitor local B cell activation. Objectives To evaluate the potential role of CXCL13 as biomarker of SG pathology in two independent SS cohorts. Methods 109 patients with SS were recruited at Sapienza University of Rome (Italy) (n=60), or at Queen Elizabeth Hospital in Birmingham and Barts Health NHS Trust in London (n=49). Both sera and matched paraffin-embedded minor SG biopsy were available. Sicca (n=57) and healthy subjects (HS) (n=19) sera were used as control. CXCL13 gene expression was also assessed in 25 frozen SGs. Results CXCL13 serum levels were higher in SS patients [90.3 (84.2) pg/ml], compared to both sicca [61.9 (38.6) pg/ml), p=0.0005] and HS [36.5 (40.18) pg/ml, p Conclusions Our data foster the use of CXCL13 to monitor the extent of local pathology in SS and its validation in longitudinal clinical studies. Both serum and tissue expression of CXCL13 correlate with SS histological severity, suggesting a major role of this chemokine in SG lymphocytes recruitment and organization. Disclosure of Interest None declared.

Career situation of first and presenting author Post-doctoral fellow. Introduction The development of the B cell repertoire is regulated by the process of affinity maturation that occurs within the inner part of the B cell follicles within secondary lymphoid organs (SLOs). In autoimmunity process might occur in ectopic lymphoid structures (ELS), aggregates of lymphocytes that form in target organs of disease (i.e. the salivary glands of patients with Sjogren’s Syndrome (SS)). The phenotypical and functional features supporting ELS pathogenic properties have not been identified. Moreover, the functional proof that ELS independently from SLOs contribute to the autoimmune response has not been provided. Objectives To characterise the transcriptome profile of human ELS isolated from SS salivary glands in comparison with SLO and to dissect, in an animal model of ELS development, the ability of ELS to contribute to the autoreactive response. Methods Frozen salivary gland biopsies were obtained from SS patients and selected for presence of germinal center +ELS. Samples were stained and microdissected, RNA isolated and transcribed and used for RNAseq using ClonTech SMARTseq v4 kit. Salivary gland ELS and autoimmunity was established as previoully described1 in FAP-DTR mice. Animals were locally treated (day 2 and 6) with DTX to induce selective ELS disaggregation; glands and serum were harvested for analysis at day 15. Results Transcript analysis provide evidence that the ectopic ELS are characterised by upregulation of TNF, INFg, BAFF, APRIL, CXCL12 and CXCL13, FAS and FASL as compared to SLOs. Sequencing unveiled an alterated cell-proliferation profile with downregulation of BCL6 and AID, the enzymes responsible for B cell affinity maturation. Selective depletion of FAP +cells induced loss of anatomical segregation of ELS and profoundly compromised their anatomy with significant impact on the local autoimmune response. Conclusions Our study provides the first evidence of a significant molecular difference between ELS and SLOs. We demonstrated that, although characterised by similar anatomical organization, ELS pathogenic cytokine signature is associated with low levels of bcl6 and AID and aberrant apoptosis, unveiling impaired regulation of the B cell cycle, responsible for the survival of autoreactive, poorly selected B cell clones. Accordingly, selective depletion of ELS in vivo profoundly impact local and systemic autoimmunity. Reference Barone F, et al. PNAS 2015. Disclosure of Interest None declared.

Sjogren’s syndrome (SS) is a chronic autoimmune disease characterised by B-cell hyper-activation and exocrine gland infiltration that results in loss of glandular function, systemic manifestations and autoantibody production. The phosphatidylinositol 3–kinase delta isoform (PI3Kδ) belongs to a family of intracellular lipid kinases that regulate metabolism, survival, proliferation, apoptosis, growth, and cell migration and has been successfully targeted in B-cell malignancies. Given the central role of the B-cells in the pathogenesis of SS we investigated evidence for the engagement of the PI3Kδ pathway in SS and the functional consequences of blocking PI3Kδ in an animal model of SS. PI3K pathway activation was investigated in salivary gland (SG) biopsies from patients with SS or non-specific chronic sialoadenitis (NSCS). Samples were stained for phosphorylated ribosomal protein S6 (pS6), a downstream effector in the PI3Kδ pathway. UCB5857, a small molecule inhibitor of PI3Kδ, was used either prophylactically or therapeutically in vivo in an inducible model of ectopic lymphoneogenesis in murine SGs that mimics SS. Flow cytometry, immunofluorescence and quantitative PCR was used on the murine isolated SG to evaluate the samples at peak of inflammation which is day 15 post-cannulation. Histological staining for pS6 showed significant expression of pS6 in SS samples relative to NSCS control, confirming engagement of the PI3K pathway. pS6 was detected within lymphoid aggregates in both T- and B-cell areas and on the periphery of the lymphoid foci in SG biopsies. pS6 staining was predominantly found on CD138 + plasma cells in SG biopsies. Mice treated either prophylactically or therapeutically with UCB5857 displayed decreased lymphocyte infiltration in cannulated salivary glands relative to vehicle treated mice. Additionally, a gene expression profile associated with ectopic lymphoneogenesis (CXCL13, CCL19, CCL21) was significantly impaired in mice treated with UCB5857. Lymphoid aggregates in these mice were characterised by decrease in focus score, smaller size and reduced T/B-cell follicular organisation. Preliminary data implicates activation of PI3Kδ pathway in several cells within the SGs of SS patients that may contribute towards pathology. Accordingly, prophylactic and therapeutic blocking of PI3Kδ results in disaggregation of the inflammatory foci and resolution of SG inflammation in an animal model of SS.

Background:Sjögren’s Syndrome (SS) is characterized by chronic inflammation supported by intrinsic activation of salivary gland epithelial cells (SGECs). Eventually, apoptosis of SGECs ensues, which leads to salivary gland dysfunction and exposition of autoantigens. Autophagy is a stress coping mechanisms of cells implicated in both survival and exposition of autoantigens, and is thereby plausibly implicated in the pathogenesis of SS. At present, the exact relationship between apoptosis and autophagy in SS SGECs is unclear, as is the link between these mechanisms and SGECs activation.Objectives:To explore autophagy in SGECs from patients with SS and to evaluate its relationship with apoptosis and SGECs activation.Methods:Consecutive patients with suspected SS referring to our “Sjogren Clinic” were enrolled, and minor salivary gland (MSG) biopsies were collected for: (1) SGECs culture, (2) PCR analysis, (3) IFI analysis. In SGECs cultures, the expression of autophagy (LC3II), apoptosis (annexin V/PI) and adhesion molecules (ICAM) was investigated by flow cytometry (results expressed as mean % ± SD). The expression of the autophagy gene MAP1LC3II was evaluated by PCR (expressed as 2^deltaCT normalized to GADPH) on both MSG sections and MSG acinar and ductal epithelium samples obtained by laser capture microdissection. Tissue expression of LC3II was evaluated by IFI on SS MSG.Results:Primary SGECs cultures were established from 14 MSG obtained for diagnostic purposes (SS n=8, Sicca n=6). These cells exhibited an inverse correlation between apoptosis and autophagy (p=0.007, r=-0.784), with lower levels of apoptosis (19.7±6.5 vs 24.5±8.5, p=ns) and higher levels of autophagy (59.7±13.1 vs 54.19±19.4, p=ns) in SS compared to Sicca. In SS, MAP1LC3 was positively correlated with Focus Score (p=0.021 r=0.478); however, PCR studies did not reveal significant differences in MAP1LC3 expression between SS (n=26) and Sicca (n=15) (0.024±0.010 vs 0.022±0.008, p=ns). Ductal SGECs (n=4) isolated by laser microdissection of MSG revealed a higher expression of MAP1LC3 (0.005±0.0005 vs 0.003±0.0008; p=0.057) compared to normal acinar epithelium (n=5); a major expression of LC3II in ducts was confirmed by IFI (Image).In SS, a higher expression of ICAM compared to sicca was observed (11.1±3.8 vs 6.9±6.9, p=0.006) and autophagy and apoptosis showed a trend of positive and negative correlation with this molecule, respectively (p=0.683 r=0.118 and p=0.106 r=-0.446).Figure.LC3-II staining in SS MSG [LC3-II+ (green) and Hoechst stain (blue); 60x magnification].Conclusion:In SS, autophagy is upregulated in SGECs and inversely correlated with apoptosis, thus supporting a role of this process in cells’ death prevention during inflammatory process. Indeed, the degree of msg inflammation is correlated more with the activation of autophagy than apoptosis. Interesting, in SS, SGECs autophagy is mainly observed at ductal level and is correlated with higher expression of adhesion molecules suggesting a link between this pathway and changes in SGECs immune phenotype.Disclosure of Interests: :Serena Colafrancesco: None declared, cristiana barbati: None declared, Valentina Iannizzotto: None declared, Linda Mastromanno: None declared, Saba Nayar: None declared, Elena Pipi: None declared, angelica gattamelata: None declared, francesco ciccia Grant/research support from: pfizer, novartis, roche, Consultant of: pfizer, novartis, lilly, abbvie, Speakers bureau: pfizer, novartis, lilly, abbvie, cristiano alessandri Grant/research support from: Pfizer, Francesca Barone: None declared, fabrizio conti Speakers bureau: BMS, Lilly, Abbvie, Pfizer, Sanofi, Roberta Priori: None declared

Objectives SS is an autoimmune condition characterized by systemic B-cell activation, autoantibody production and ectopic germinal centres’ formation within the salivary gland (SG). The extent of SG infiltrate has been proposed as a biomarker of disease severity. Plasma levels of CXCL13 correlate with germinal centres’ activity in animal models and disease severity in SS, suggesting its potential use as a surrogate serum marker to monitor local B-cell activation. The aim of this study was to evaluate the potential role of CXCL13 as a biomarker of SG pathology in two independent SS cohorts. Methods 109 patients with SS were recruited at Sapienza University of Rome (Italy) (n = 60), or at Queen Elizabeth Hospital in Birmingham and Barts Health NHS Trust in London (n = 49). Both sera and matched minor SG biopsy were available. Sicca (n = 57) and healthy subjects’ (n = 19) sera were used as control. Results CXCL13 serum level was higher in SS patients compared with controls. Correlations between its serum levels and a series of histomorphological parameters, including size of the aggregates and the presence germinal centres', were observed. Conclusion Our data foster the use of CXCL13 to monitor the extent of local pathology in SS and its validation in longitudinal clinical studies.

Background Salivary glands of primary Sjogren’s syndrome (pSS) are characterised by complex leukocyte infiltration organised into tertiary lymphoid structures (TLS). The mechanisms regulating leukocyte trafficking into inflamed salivary glands are poorly described, but dysregulated T-cell recruitment during inflammation is believed to contribute to disease onset and chronicity. We recently described a homeostatic pathway in which a B cell-derived peptide (PEPITEM), secreted in response to adiponectin, regulates T-cell trafficking during inflammation via sphingosine-1-phosphate activity on endothelial cells1. Disruption of this pathway by downregulation of adiponectin receptors on circulating B cells has been demonstrated in type-1 diabetes and rheumatoid arthritis, suggesting a potential role for PEPITEM in the pathogenesis of autoimmune diseases, and indicating a role for adiponectin receptors as biomarkers in these conditions1. Objectives We aimed to investigate the efficacy of PEPITEM as an inhibitor of T-cell trafficking in an inducible animal model of salivary gland inflammation that mimics the histological features of pSS, and to investigate the potential translatability of this pathway in patients with pSS. Methods Submandibular salivary glands of C57BL/6 mice were intra-ductally cannulated with luciferase-encoding replication-deficient adenovirus to induce TLS formation as previously described2. Mice were administered daily either PBS or PEPITEM by intraperitoneal injection from day 0, and their salivary glands dissected at day 5 post cannulation. T-cell infiltration into salivary glands was assessed using a combination of flow cytometry, immunofluorescence, and qRT-PCR for inflammatory chemokines. Results B cells in sera from cannulated animals express lower levels of both adiponectin receptors 1 and 2 in comparison with non-inflamed control mice. In cannulated animals treated with PEPITEM, histological analysis of salivary glands revealed fewer, as well as less aggregated, infiltrating T cells. Both CD4 +and CD8+numbers were significantly lower in the salivary glands of PEPITEM-treated animals. Furthermore, administration of PEPITEM also decreased mRNA transcripts for lymphotoxin beta, IL-7, lymphoid chemokines (CCL19 and CXCL13) and T-cell chemokine receptor CCR7, cytokines and chemokines known to regulate ectopic lymphoneogenesis in pSS. Samples from pSS patients are currently being assessed to validate the relevance of this pathway in pSS. Conclusions These results demonstrate that administration of exogenous PEPITEM can reduce T-cell influx into salivary glands. This may represent a rescue of the homeostatic regulation of leukocyte trafficking which is disrupted in inflammation. Our work suggests that PEPITEM should be considered to address therapeutic needs in chronic inflammatory conditions, and that the detection of decreased levels of adiponectin receptors could serve as a biomarker in pSS. References [1] Chimen, McGettrick, et al. Nat Med. 2015;21(5):467–475. [2] Bombardieri, Barone, et al. J. Immunol. 2012;189:3767–3776. Disclosure of Interest None declared

Introduction The RANKL (ligand)-RANK-OPG triad, members of the TNF(R) superfamily, is implicated in lymphoid organ development and bone homeostasis. It has recently been demonstrated that RANK-activated astrocytes release CCL20 and attract T cells to the central nervous system in a model of Multiple Sclerosis and that transgenic RANK expression in the skin promotes aberrant epithelial cell proliferation and is sufficient to induce ectopic formation of tertiary lymphoid structures (TLS). Ductal epithelial cells (SGEs) have been implicated in Sjogren’s Syndrome (SS) pathogenesis where they mediate immune recruitment by expression of pro-inflammatory chemokines and support the formation of pre-malignant myoepithelial lesions. Objectives To address the role of RANK-RANKL interaction in primary (p) SS. Methods A combination of human and mouse studies were used to explore the RANK-RANKL interaction in pSS. Salivary glands (SGs) and saliva samples from patients recruited in the OASIS cohort (University of Birmingham) were studied to evaluate this pathway in human disease. Consecutive stimulated saliva samples (n=69) were analysed using Proseek Multiplex INF96 × 96, covering 92 unique inflammation-related protein biomarkers. Taking advantage of a viral induced model of pSS we studied the effect of this pathway with a RANKL blocking antibody and by inducing gain of function with direct cannulation in the salivary glands of recombinant RANKL. Murine SGs were studied by immunofluorescence, flow cytometry and qPCR on total tissue and sorted cells. Results Fourteen proteins in saliva were significantly separated between pSS and sicca controls, and elevated levels of just two proteins, RANKL and TNFβ, could classify pSS or sicca with 75% accuracy. Levels of salivary RANKL and CCL20 were strongly correlated (r=0.6; p Conclusions In vivo RANK/RANKL interaction mediates recruitment of activated T cells that are skewed toward a Th2 phenotype. These, in turn, will favour the establishment of TLS in the SG. Those data were confirmed in human pSS, where expression of RANK is found in inflamed epithelium and RANKL detection in saliva is able to differentiate patients with pSS from sicca controls, thus candidates this pathway both for drug targeting and patient stratification. Disclosure of interest None declared

TP63 is the most ancient member of the p53 gene family. The p53 family comprises three transcription factors (p53/p63/p73). They share a high degree of homology and similar domain structure. Yet, they can exist as truncated isoforms. Alternative promoters and splicing sites lead to the generation of several molecules. P53/p63/p73 are important to maintain cell homeostasis. P63 and p73 regulate many p53 target genes. This is due to their common structural features. Both proteins may compensate the loss of p53. This is a common event occurring in more than 50% of malignancies. Yet, p63 (or p73) has its own role. Studies from p63-null mice have shown the key role of p63 in embryo development. Several reports have supported the p63 role in epidermal development and in skin homeostasis. P63 involvement in heart development is currently being researched. Recent studies have found p63 to be "the guardian of human reproduction". In addition, p63 has an important, even controversial, role in cancer. Here, we provide a general overview of p63 regulation and activity. We discuss emerging concepts about its role in germ line protection, metabolism and cancer.

The tumor suppressor p53 is a transcription factor that regulates key processes. But, the outcomes of the p53 response go beyond its role as a nuclear transcription factor. Sirtuin (SIRT1) regulates p53 functions as transcription factor. At the same time, SIRT1 protects the genome under stress conditions. The link between p53 and SIRT1 responses is unique. Both regulate metabolism, stress signaling, cell survival, cell cycle control and genome stability. Recent studies have proposed cancer as a metabolic disease. This is due to the switch from aerobic to anaerobic metabolism during tumor development. Yet, the complex molecular circuits (in and out of the nucleus) of tumor progression remain elusive. In this review, we will focus on the interplay between p53 and SIRT1. We will discuss their roles as nodes for possible therapeutic intervention.