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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

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.

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