Discordant Predictions of Extraglandular Involvement in Primary Sjögren’s Syndrome According to the Anti-SSA/Ro60 Antibodies Detection Assay in a Cohort Study

Electrophoresis-derived techniques for anti-SSA/Ro60 KDa (anti-SSA) antibodies detection have been progressively replaced by methods using non-native antigens. We aimed to compare the patients’ phenotypes and the occurrence of extraglandular manifestations in primary Sjögren’s syndrome according to the method used to detect anti-SSA antibodies. Sera from patients with a diagnosis of pSS according to ACR/EULAR 2016 criteria between 2008 and 2017 were tested for anti-SSA antibodies using methods with non-native antigens (magnetic bead multiplex assay; line immunoassays) and one with native antigens (counterimmunoelectrophoresis (CIE)). The population was split into three groups according to anti-SSA antibodies status: absence (SSA−), presence in any method except for CIE (SSA+CIE−), and presence in CIE (SSA+CIE+). The patients in the SSA+CIE+ group (n = 70, 42.7%) were ten years younger and presented more immunological activity compared with both the SSA− (n = 80, 48.8%) and SSA+CIE− groups (n = 14, 8.5%). The SSA− and SSA+CIE− groups were poorly distinct. The presence of anti-SSA antibodies solely in CIE was significantly associated with the occurrence of extraglandular manifestations of pSS (HR = 4.45 (2.35–8.42)). Contrary to CIE, methods using non-native antigens to detect anti-SSA antibodies were unable to predict the occurrence of systemic expression of pSS.

Correlation of peripheral CD4+GranzB+CTLs with disease severity in patients with primary Sjögren’s syndrome

Introduction Primary Sjögren’s syndrome (pSS) is a chronic systemic autoimmune disease which has focal lymphocytic infiltration including a majority of CD4+ T cells. This study was to investigate the correlation of peripheral granzyme B (GranzB)-expressing CD4+ T cells with disease severity and histological lesion in patients with pSS. Methods We recruited 116 pSS and 46 health control (HC) using flow cytometry to examine the percentage of CD4+GranzB+CTLs in the peripheral blood, and immunofluorescence to test their expression in the labial gland. Results The percentage of CD4+GranzB+CTLs was significantly upregulated in pSS than in HC (7.1 ± 4.9% vs 3.1 ± 1.9%, p < 0.0001) and positive correlation with ESSDAI. The frequency of them was markedly higher in pSS with extraglandular manifestations. After excluding the other risk factors associated with pSS, they were still related to ESSDIA and extraglandular manifestations independently (p < 0.05), and they are the risk factor of extraglandular involvement (odds ratio = 1.928). Moreover, they could be observed in the LSGs. ROC curve analysis indicated that the area under the curve (AUC) of CD4+GranzB+CTLs was 0.796 to predict the activity of pSS and 0.851 to presume extraglandular manifestations. The best diagnostic cutoff point was 4.865 for pSS patients. Conclusion In this study, we provide new evidence indicating the involvement of CD4+GranzB+CTLs over activation in the pathophysiology of pSS, which may serve as a new biomarker to evaluate the activity and severity of pSS.

Correlation of Peripheral CD4+GranzB+CTLs with Disease Severity in Patients with Primary Sjögren’s Syndrome

Introduction: The major histopathologic lesion of SS is a focal lymphocytic infiltrate, which include a majority of CD4+T. Several studies have shown that the epithelial cells in SS present diverse phenomena, such as MHC class II overexpression. CD4+T cells with cytotoxic activity are characterized by their ability to secrete granzyme B to kill the target cells in an MHC class II-restricted fashion. So this study was to investigate the correlation of peripheral CD4+GranzB+CTLs with pSS. Methods: We recruited 116 pSS and 46 HC using flow cytometry to examine the percentage of CD4+GranzB+CTLs in peripheral blood, and immunofluorescence to test their expression in labial gland. Results: The percentage of CD4+GranzB+CTLs were significantly up-regulated in pSS than HC (7.1±4.9% vs 3.1±1.9%, p <0.0001) and positive correlation with ESSDAI. The frequency of them were markedly higher in pSS with extraglandular manifestations. After excluding the other risk factors associated with pSS, they were still related to ESSDIA and extraglandular manifestations independently(p<0.05) and they are the risk factor of extraglandular involvement (odds ratio=1.928). Moreover, They could be observed in the LSGs. ROC curve analysis indicated that the area under the curve (AUC) of CD4+GranzB+CTLs was 0.796 to predict the activity of pSS, and 0.851 to presume extraglandular manifestations. The best diagnostic cut-off point was 4.865 for pSS patients. Conclusion: In this study, We provide new evidence indicating involvement of CD4+GranzB+CTLs over activation in the pathophysiology of pSS, which may serve as a new biomarker to evaluate the activity and severity of pSS.

POS0776 CORRELATION OF PERIPHERAL CD4+GRANZB+CTLS WITH DISEASE SEVERITY IN PATIENTS WITH PRIMARY SJÖGREN’S SYNDROME

Background:Sjögren’s syndrome (SS) is a chronic autoimmune disorder. The major histopathologic lesion of it is a focal lymphocytic infiltrate around ductal and acinar epithelial cells, which include a majority of CD4+T. Several studies have shown that the epithelial cells in SS present diverse phenomena, such as MHC class II overexpression. CD4+T cells with cytotoxic activity (CD4 CTL) have been detected in various immune responses. They are characterized by their ability to secrete perforin and granzyme B to kill the target cells in an MHC class II-restricted fashion.Objectives:So this study was to investigate the correlation of peripheral CD4+GranzB+CTLs with disease severity and organ involvement in patients with primary Sjögren’s syndrome.Methods:We recruited 116 pSS patients and 46 healthy controls using flow cytometry to examine proportion of CD4+GranzB+CTLs in their peripheral blood, and immunofluorescence to test the expression of CD4+GranzB+CTLs in labial gland. The correlations of CD4+GranzB+CTLs and the relevant clinical data were analyzed.Results:We analyzed the percentage of CD4+GranzB+cytotoxic T cells in peripheral blood mononuclear cells (PBMCs) by flow cytometry. Frequency of peripheral CD4+GranzB+CTLs were measured in 116 patients with pSS and 46 healthy controls matched for age and sex. The percentage of CD4+GranzB+CTLs were significantly up-regulated in pSS patients than healthy controls (7.1%±4.9% vs 3.1%±1.9%, p <0.0001) and positive correlation with ESSDAI in pSS patients(r = 0.6332, p<0.001). The percentage of CD4+GranzB+CTLs were markedly higher in pSS patients with extraglandular manifestations. Moreover, CD4+GranzB+CTLs were observed in the lymphocytic foci and periductal areas of the LSGs and were elevated with increased foci index (FI). After excluding the other risk factors associated with pSS, CD4+GranzB+CTLs were still related to ESSDIA and extraglandular manifestations independently(p<0.05). ROC curve analysis indicated that the area under the curve (AUC) of CD4+GranzB+CTLs was 0.796 to predict the activity of pSS, and 0.851 to presume extraglandular manifestations. The best diagnostic cut-off point was 4.865 for pSS patients.Conclusion:In this study, We provide new evidence indicating involvement of CD4+GranzB+CTLs over activation in the disease pathophysiology of pSS, which may serve as a new biomarker to evaluate the activity and severity of pSS.References:[1]Takeuchi A, Saito T. Front Immunol. (2017) 23:194.[2]Brown DM, et al. Front Immunol. (2016) 9:93.[3]Polihronis M, et al. Clin Exp Immunol. (1998) 114:485-90.[4]Xanthou G, et al. Clin Exp Immunol. (1999) 118:154-63.[5]Maehara T, et al. Ann Rheum Dis. (2017) 76:377-385.[6]Goules AV, et al. Clin Immunol. (2017) 182:30-40.[7]Hashimoto K, et al. Proc Natl Acad Sci U S A. (2019) 116:24242-24251.[8]Croia C, et al. Arthritis Rheumatol. (2014) 66:2545-57.[9]Schmidt D,et al. J Clin Invest. (1996) 97:2027–37.[10]Pandya JM, et al. Arthritis Rheum. (2010) 62:3457–66.[11]Moosig F, et al. Clin Exp Immunol. (1998) 114:113–8.[12]Peeters LM, et al. Front Immunol. (2017) 20:1160.Table 1.Multivariate analysis of CD4+GranzB+CTLs influenced by pSS-related factorsregression coefficientstandard errort-statisticsp value95%CICD8+GranzB+CTLs(%)0.1440.0334.3346.9E-50.077, 0.211ESSDAI0.2560.1222.0950.0410.011, 0.502extraglandular manifestations2.6121.2682.0590.0450.065, 5.158Figure 1.Receiver operating characteristic (ROC) curve of the frequency of CD4+GranzB+CTLs to predict ESSDAI and extraglandular manifestations responseDisclosure of Interests:None declared

Atypical Sjögrenʼs Syndrome Initially Presenting as Lymphocytic Interstitial Pneumonitis followed by Immune Thrombocytopenia

Background. Sjögrenʼs syndrome is an autoimmune disease characterized primarily by decreased exocrine gland function leading to eye and mouth dryness. Extraglandular manifestations occur less frequently. Case Report. A 74-year-old man with hypertension was admitted with productive cough and fever. On physical examination, he had bilateral lower lung decreased breath sounds. A chest radiograph showed bibasilar patchy infiltrate. Laboratory studies revealed hemoglobin of 11.9 g/dL, white blood cell count of 16,000/uL, and platelet count of 250,000/uL. Empiric antibiotic therapy was begun for suspected community acquired pneumonia, and then he was discharged home. However, his cough recurred. Chest computed tomography demonstrated adenopathy throughout the mediastinum and multiple ill-defined patchy groundglass opacities with a lower lobe prominence. He underwent a transbronchial biopsy to rule out malignancy; however, it showed lymphocytic interstitial pneumonitis. Antinuclear antibody was 1 : 80 homogeneous, and anti-SSA antibody was 6.3 AI (normal <1.0 AI). The patient was treated with prednisone 20 mg/day with marked improvement in his symptoms. Repeat chest computed tomography showed decreased groundglass opacities and decreased mediastinal lymph nodes. After more than a year, he was readmitted due to petechiae on his buccal mucosa and a platelet count of 2000/μL. The patient was started on prednisone 80 mg/d and intravenous immunoglobulin 80 g/d for 2 consecutive days. The platelet count eventually increased to 244,000/μL. Conclusion. We report a rare presentation of Sjogrenʼs syndrome manifesting as acute lymphocytic interstitial pneumonitis and followed by immune thrombocytopenia. Both extraglandular manifestations responded well to corticosteroid therapy.

Characteristics of clinical, laboratory, and immunological manifestations in patients with anticentromere antibody-associated Sjögren's disease

Objective: to study clinical and laboratory features in patients with anticentromere antibody (ACA)-positive SjЪgren's disease (SD), as well as the sensitivity of different methods for determination of ACA, and to elaborate an algorithm for differential diagnosis in ACA-positive patients.Patients and methods. The V.A. Nasonova Research Institute of Rheumatology followed up 136 patients who were highly positive for ACA. The investigators used the 2001 Russian criteria for the diagnosis for SD; the 2013 ACR/European League Against Rheumatism (EULAR) criteria for that of scleroderma systematica (SDS); the guidelines of the American Association for the Study of Liver Diseases, the Russian Gastroenterological Association, and the Russian Society for the Study of the Liver for that of primary biliary cholangitis (PBC)/biliary duct epitheliitis in the presence of SD. Lymphomas were diagnosed by biopsies of affected organs according to the WHO classification. SD was diagnosed in 119 patients; SDS in 49 cases (37 with SDS concurrent with SD and 12 with isolated SDS), PBC/biliary duct epitheliitis in 23 (all cases with PBC/biliary duct epitheliitis concurrent with SD and/or SDS); 5 patients were excluded from the investigation. Further analysis included 131 ACA-positive patients. The patients were divided into three groups: SD (n=82 or 62.6%); SD+SDS (n=37 or 28.24%); SDS (n=12 or 9.16%).Results and discussion. Autoantibodies to centromere peptide (CENP) A and CENP-B in the same titers were detected in all ACA-positive patients, regardless of diagnosis. Comparative analysis of three patient groups revealed no statistically significant differences in the frequency of laboratory deviations. The signs characteristic of classical SD (rheumatoid factor (RF)), anti-Ro and anti-La antibodies, leukopenia, higher ESR values, hypergammaglobulinemia, and elevated IgG/IgA levels) were found in a small proportion of patients. The frequency and severity of glandular manifestations did not differ in SD and SD + SDS. PBC/biliary duct epitheliitis was present in 17.5% of ACA-positive patients (in most antimitochondrial antibody-positive cases); no statistically significant differences in its frequency were found between the groups. Other extraglandular manifestations in SD and SD + SDS were identified in a smaller number of patients. All sclerodermic spectrum manifestations were more common in SD and SD + SDS than in BS. Pulmonary arterial hypertension was not diagnosed in any patient from the SD group. MALT lymphomas were detected in 19 ACA-positive patients. Those were present only in BS patients and absent in the SDS group. MALT lymphomas developed in the first 10 years after the onset of SD. The transformation of MALT lymphoma into diffuse large B-cell lymphoma was observed in 2 patients. The main signs of lymphomas in SD patients were persistent parotid salivary gland enlargement, decreased levels of complement C4 and peripheral blood CD19+ cells, as well as cryoglobulinemic vasculitis, serum monoclonal secretion, lymphoid infiltration in the minor salivary glands (a focus score of >4), and severe damage to the salivary and lacrimal glands.Conclusion. ACA-associated SD is an independent disease subtype characterized by an increased risk for SDS, PBC, and MALT lymphomas and by a low frequency of the systemic manifestations and laboratory signs characteristic of classical SD. Regardless of the detected type of antibodies and the presence or absence of extraglandular manifestations, damage to the salivary and lacrimal glands progresses in SD, which often leads to lymphomas; therefore, the therapy that may prevent this complication should be initiated as soon as possible after SD diagnosis. The lymphoproliferation signs identified in this investigation should be taken into account in all ACA-positive patients with SD for the early diagnosis of lymphoid tumors before therapy is prescribed. An algorithm for differential diagnosis in seropositivity for ACA is presented. Determination of autoantibodies to CENP-A and CENP-B does not allow the differential diagnosis in ACA-positive patients.

FRI0161 PHENOTYPIC DIFFERENCES BETWEEN SJÖGREN’S SYNDROME PATIENTS WITH LOW AND HIGH-GRADE INFLAMMATION BASED ON SALIVARY GLAND FOCUS SCORE

Background:Sjögren’s syndrome (SS) is characterized by the presence of lymphocytic infiltration around the ductal epithelium of the salivary and lachrymal glands. The periepithelial inflammatory lesions and the enclosed B cell component are responsible for the glandular and extraglandular manifestations of the disease. Previous studies have shown that the severity of inflammation observed within the salivary glands is correlated with the occurrence of extraglandular manifestations. However, in these studies either the number of patients is small or the SS criteria are not well defined. To explore the association between the degree of inflammation within the salivary glands and the phenotype of the disease, large and well characterized cohorts of SS patients is required.Objectives:To compare the phenotypic features of SS patients with low and high degree of inflammation within the minor salivary glands as reflected by the focus score (FS).Methods:From a total cohort of 1723 consecutive SS patients who fulfill the 2016 EULAR/ACR criteria and are followed up in 4 clinical centers ([Universities ofPisa,Athens,Harokopio andIoannina, (PAHI)], those who had performed a lip biopsy and the focused score was available, were classified into low grade (FS<3) or high grade (FS≥3). Glandular (dry mouth, dry eyes, parotid gland enlargement) and extra-glandular manifestations (Raynaud’s phenomenon, arthralgias/myalgias, arthritis, palpable purpura, liver involvement, kidney involvement, lung involvement, neurologic involvement, long standing lymphadenopathy and lymphoma) as well as serologic features (ANA, RF, anti-Ro/SSA, anti-La/SSB) were compared between the 2 groups. Statistical analysis for categorical variables was performed by Fisher exact or chi-square tests and for continuous variables with t test or Mann-Whitney accordingly.Results:Eight hundred and eight minor salivary gland biopsies were available and evaluated based on focus score at the initial evaluation of SS patients, of whom 753 had low grade (FS<3) and 153 high grade (≥3) inflammation. The median disease duration after SS diagnosis was not statistically significant different for the 2 groups (median: 4 years, range: 0-36 years). SS patients with high grade inflammation displayed higher prevalence of salivary gland enlargement (SGE) (40% vs 25%, p=0,0002), long standing lymphadenopathy (22% vs 14%, p=0,02), ANA (97% vs 88%, p=0,0001), anti-La/SSB (52% vs 32%, p<0,0001), RF (61,5% vs 48%, p=0,003), peripheral neuropathy (PN) (5,3% vs 1,5, p=0,01) and of lymphoma (26% vs 8%, p<0,0001, OR=4,142, 95%CI=2,65 to 6,47) compared to those with low grade inflammation.Conclusion:SS patients with FS ≥3 at the initial evaluation, display higher prevalence of lymphoma as well as higher B cell hyperactivity and certain clinical manifestations (SGE, PNS, lymphadenopathy) that constitute risk factors for lymphoma development.Disclosure of Interests:Loukas Chatzis: None declared, Vasileios Pezoulas: None declared, Francesco Ferro: None declared, Valentina Donati: None declared, Aliki Venetsanopoulou: None declared, Evangelia Zampeli: None declared, Maria Mavromati: None declared, Paraskevi Voulgari: None declared, Clio Mavragani: None declared, Dimitris Fotiadis: None declared, Fotini Skopouli: None declared, Salvatore De Vita Consultant of: Roche, Human Genome Science, Glaxo Smith Kline and Novartis, Gorgoulis Vassilis: None declared, Chiara Baldini: None declared, Haralampos M. Moutsopoulos: None declared, Andreas Goules: None declared, Athanasios Tzioufas: None declared

Minor salivary gland biopsy: its importance in rheumatoid arthritis and secondary Sjögren's síndrome

Secondary Sjögren’s Syndrome (SSs) is common in patients with Rheumatoid Arthritis (RA). Objectives: to determine if lymphocytic sialoadenifitis (FLS) is associated with clinical and serological differences in a group of patients with RA and SSs. Methods: patients with a diagnosis of RA and SSs were included, which of them presented FLS in the salivary gland biopsy, clinical and serological characteristics were compared. Results: 88 patients were included, 92% women, mean age of 53 years (SD ± 11.3) and 12.5 years of evolution of RA (RIC 6-7). 63.6% had SLF versus 36.4% who did not. In the univariate analysis, a statistically significant association was found between FLS + and the variables: parotidomegaly, interstitial lung disease, hypergammaglobulinemia, hypocomplementemia, rheumatoid factor, positive ANF, and extra-articular and/ or extraglandular manifestations. In the multivariate analysis, the variables independently associated with the presence of FLS were: extra-articular and/or extraglandular manifestations (OR 5.67, 95% CI 1.6-20), positive ANF (OR 11.7, 95% CI 1.6-83) and hypergammaglobulinemia (OR 21, 95% CI 2.46-179). Conclusion: patients with RA and SSs with FLS have a higher frequency of extra-articular and extraglandular manifestations and serological differences, which would imply a different clinical follow-up.