Objective Sjögren’s disease (SjD) is a chronic exocrine disorder typified by inflammation and dryness, but also profound fatigue, suggesting a pathological basis in cellular bioenergetics. In healthy states, dysfunctional mitochondria are recycled by mitophagic processes; when impaired, poorly functioning mitochondria persist and produce inflammatory reactive oxygen species. Employing a case–control study, we tested our hypothesis that mitochondrial dysregulation in T cells is associated with fatigue in SjD.Methods We isolated pan T cells from peripheral blood mononuclear cells of 13 SjD and 4 non-Sjögren’s sicca (NSS) subjects, who completed several fatigue questionnaires, along with 8 healthy subjects. Using Seahorse, we analysed T cells for mitochondrial oxygen consumption rate (OCR) and extracellular acidification rate, which we assessed for correlation with fatigue measures. Using public microarray data available for 190 SjD and 32 healthy subjects, we identified a mitophagic transcriptional signature that stratified SjD patients into 5 discrete clusters. Comparisons between the SjD subjects in these clusters to healthy individuals identified differentially expressed transcripts, which we subjected to bioinformatic interrogation.Results Basal OCR, ATP-linked respiration, maximal respiration and reserve capacity were significantly lower in SjD and NSS subjects compared with healthy individuals, with no differences in non-mitochondrial respiration, basal glycolysis or glycolytic reserve. Scores related to a sleep questionnaire and Bowman’s Profile of Fatigue and Discomfort showed correlation with altered OCR in SjD. Subgroup differential expression analysis revealed dynamic transcriptional activity between mitophagy subgroups, expanding the number of differentially expressed transcripts tenfold.Conclusions Mitochondrial dysfunction and fatigue are significant problems in SjD warranting further investigation.

BACKGROUND:Sjögren's disease is characterised by mucosal dryness, fatigue, chronic pain, systemic organ involvement, and elevated autoreactive IgG antibodies. There are no approved disease-modifying treatments. Therefore, we aimed to evaluate nipocalimab, a neonatal Fc receptor blocker that reduces circulating IgG, including autoantibodies, in patients with Sjögren's disease. METHODS:This phase 2, double-blind, multicentre trial enrolled individuals with moderate-to-severe, active Sjögren's disease (ie, Clinical European League Against Rheumatism Sjögren's Syndrome Disease Activity Index [ClinESSDAI] of at least 6) who were seropositive for anti-Ro IgG autoantibodies. Participants were recruited from 69 centres across France, Germany, Italy, Japan, the Netherlands, Poland, Portugal, Spain, Taiwan, and the USA. These centres included rheumatology centres, hospitals, and clinical research centres with experience conducting pharmaceutical company-sponsored phase 2 and phase 3 studies, which reported an ability to enrol eligible patients. Central randomisation assigned participants to one of three treatment groups using an Interactive Web Response System. Randomised (1:1:1) participants received intravenous nipocalimab 5 mg/kg, intravenous nipocalimab 15 mg/kg, or placebo every 2 weeks for 22 weeks. Schedules for administering the study intervention were the same across treatment groups, and labels on the study interventions were prepared by an unmasked pharmacist and were identical to maintain masking for the participants, investigators, site staff, and sponsor. The primary endpoint was change from baseline in ClinESSDAI score at week 24. The primary endpoint and other efficacy and safety analyses included participants who were randomly assigned and who received at least one dose of study intervention. For the primary endpoint, data from the time of discontinuation and onward were considered missing. The primary analysis approach used a mixed model for repeated measures to estimate the average outcome, taking into account the non-missing data and variability. DAHLIAS was registered with EudraCT (2021-000665-32) and ClinicalTrials.gov (NCT04968912) and has been completed. FINDINGS:163 participants were recruited between Sept 21, 2021, and April 3, 2023, (53 participants to nipocalimab 5 mg/kg, 54 to nipocalimab 15 mg/kg, and 56 to the placebo). The mean age of participants was 48·1 years (SD 12·12); 151 (93%) participants were female and 12 (7%) were male. The nipocalimab 15 mg/kg group had a significant reduction in ClinESSDAI score at week 24 versus the placebo group (least squares mean difference -2·65, 90% CI -4·03 to -1·28; p=0·0018), and the nipocalimab 5 mg/kg group had a non-significant reduction versus placebo (-0·34, -1·71 to 1·03; p=0·68). The safety profile of nipocalimab was comparable, for both doses, with that of placebo, with generally similar rates of adverse events and serious adverse events across groups. INTERPRETATION:Fc receptor blockade by nipocalimab 15 mg/kg significantly improved clinical disease activity versus placebo and was safe and well tolerated in participants with moderate-to-severe, active Sjögren's disease. Reductions in IgG autoantibodies during nipocalimab treatment support their contribution to Sjögren's disease pathogenesis. FUNDING:Johnson & Johnson.

OBJECTIVES:Diagnosis of Sjögren's disease (SjD) consists of clinical examinations that include invasive studies such as lower lip biopsies and blood collection to identify presence of serum autoantibodies. Salivary glands of patients with SjD are sites where antibody-secreting cells accumulate and secrete immunoglobulins. Many patients with dry manifestations who do not meet classification criteria are grouped as non-Sjögren's sicca (NSS) and are heavily understudied. We undertook this cross-sectional observational study to investigate the role of salivary autoantibodies as a diagnostic tool and determine presence of salivary autoantibodies in patients with NSS. METHODS:In this cross-sectional observational study, we screened whole unstimulated saliva from 446 subjects by direct enzyme-linked immunosorbent assays and capillary western blotting for anti-Ro60, anti-La, and rheumatoid factor (immunoglobulin (Ig)G and IgA) antibodies. All subjects were classified following the ACR/EULAR classification at the Oklahoma Medical Research Foundation Sjögren's Research Clinic. RESULTS:Patients with SjD were significantly more likely to have salivary antibodies compared with those with NSS and healthy control subjects. In this cohort, there were 88 subjects with NSS with seronegative profiles who had detectable salivary autoantibodies and objective measures of dryness. CONCLUSIONS:Of these 88 subjects with NSS, we identified 75 subjects that had positive objective examinations of dryness and could belong to an early-onset SjD group. Alternatively, these 75 subjects with NSS could belong to a distinct phenotype of SjD that will remain seronegative while being saliva positive for anti-Ro60. These data showed a relationship between ocular and oral dryness and the presence of salivary anti-Ro60 antibodies in subjects with NSS.

Fine mapping and bioinformatic analysis of the DDX6-CXCR5 genetic risk association in Sjögren's Disease (SjD) and Systemic Lupus Erythematosus (SLE) identified five common SNPs with functional evidence in immune cell types: rs4938573, rs57494551, rs4938572, rs4936443, rs7117261. Functional interrogation of nuclear protein binding affinity, enhancer/promoter regulatory activity, and chromatin-chromatin interactions in immune, salivary gland epithelial, and kidney epithelial cells revealed cell type-specific allelic effects for all five SNPs that expanded regulation beyond effects on DDX6 and CXCR5 expression. Mapping the local chromatin regulatory network revealed several additional genes of interest, including lnc-PHLDB1-1. Collectively, functional characterization implicated the risk alleles of these SNPs as modulators of promoter and/or enhancer activities that regulate cell type-specific expression of DDX6, CXCR5, and lnc-PHLDB1-1, among others. Further, these findings emphasize the importance of exploring the functional significance of SNPs in the context of complex chromatin architecture in disease-relevant cell types and tissues.

Background: Sjögren's disease (SjD) is a chronic, systemic autoimmune disease characterized by the presence of specific autoantibodies (AAb) and lymphocytic infiltration of exocrine glandular tissues. The pathophysiology of SjD results from dysregulated humoral immunity involving aberrant B-lymphocyte activity, leading to abnormally elevated immunoglobulin G (IgG) and IgG AAb levels, particularly anti-Ro and anti-La, which are associated with disease severity. Nipocalimab is an anti-neonatal Fc receptor (FcRn) monoclonal antibody that reduces circulating IgG, including AAb, by selectively blocking the interaction of IgG with FcRn. In phase 2 studies in AAb/alloantibody-mediated diseases, nipocalimab treatment resulted in rapid, deep, dose-dependent, reversible reductions in serum total IgG and AAb, suggesting therapeutic potential in other AAb-associated diseases, including SjD. Objectives: To evaluate the efficacy and safety of nipocalimab in patients with primary SjD. Methods: A phase 2 study (DAHLIAS; NCT04968912) was conducted in adults (18-75 years) with moderately-to-severely active primary SjD (total Clinical European League Against Rheumatism Sjögren's Syndrome Disease Activity Index [clinESSDAI] ≥6) who were seropositive for anti-Ro60 and/or -Ro52 IgG antibodies. Randomized patients (1:1:1) received IV nipocalimab at 5 or 15 mg/kg, or placebo every 2 weeks (wks) through Wk 22 and protocol-permitted background standard of care. The primary endpoint of change from baseline in clinESSDAI score at Wk 24 was chosen to avoid mechanistic bias with IgG levels. Safety assessments were conducted through Wk 30. Results: In total, 163 patients were enrolled (nipocalimab: 5 mg/kg, n=53; 15 mg/kg, n=54; placebo, n=56). Baseline characteristics were comparable between groups. The nipocalimab 15 mg/kg group met the primary endpoint versus placebo (least squares mean difference for 15 mg/kg, –2.65; 90% CI, –4.03, –1.28; p=0.002; for 5 mg/kg: –0.34; 90% CI, –1.71, 1.03; p=0.681; Table 1). Similar improvements in the 15 mg/kg nipocalimab group versus placebo were observed in most secondary/exploratory endpoints (Table 1), including change from baseline in Physician Global Assessment of Disease Severity at Wk 24 (PhGA), change from baseline in ESSDAI score at Wk 24, treatment response according to Sjögren's Tool for Assessing Response (STAR), Composite of Relevant Endpoints for Sjogren's Syndrome (CRESS), and disease activity level (DAL) response (a decrease in ≥1 clinESSDAI domain), among others. Patient-reported outcome measures were numerically improved in the 15 mg/kg nipocalimab group versus placebo. Efficacy outcomes in the 5 mg/kg nipocalimab group were generally similar to placebo. Serious adverse events were reported in 7.5%, 7.4%, and 5.4% of participants with nipocalimab 5 mg/kg, 15 mg/kg, and placebo, respectively (Table 2). Significant nipocalimab dose-dependent reductions in IgG and AAb were observed. Severe infections or infections requiring IV anti-infectives occurred in 3.8%, 1.9%, and 1.8% of participants, respectively, without a clear correlation with IgG nadir; none were deemed related to study treatment. At Wk 24, changes from baseline in albumin, low-density lipoprotein cholesterol, and total cholesterol were reported at –6.9%, 6.6% and 8.3%, respectively. No opportunistic infections or severe hypoalbuminemia were observed; no deaths were reported. Conclusion: DAHLIAS is the first study of a FcRn blocker in SjD and shows that nipocalimab treatment led to significant improvement over placebo in clinESSDAI and similar trends in other key efficacy endpoints at Wk 24 and is well-tolerated. These results demonstrate the mechanistic relevance of FcRn inhibition in SjD, indicating the potential pathogenicity of IgG AAb. Together, these findings establish proof of concept for nipocalimab in SjD and support further evaluation in patients with moderate-to-severe AAb-positive SjD. REFERENCES: NIL. Acknowledgements: NIL. Disclosure of Interests: Jacques-Eric Gottenberg AbbVie, Janssen, BMS, UCB, MSD, Novartis, Pfizer, Gilead, Galapagos, Lilly, and Sanofi, Kathy Sivils owns shares/stock options of Johnson & Johnson, Employee of Janssen, Kim Campbell owns shares/stock options of Johnson & Johnson, Employee of Janssen, Jada Idokogi owns shares/stock options of Johnson & Johnson, Employee of Janssen, Kim Hung Lo Employee of Janssen, Sophia Liva Owns shares/stock options of Johnson & Johnson, Employee of Janssen, Harman Dhatt owns shares/stock options of Johnson & Johnson, Employee of Janssen, Jonathan Hubbard owns shares/stock options of Johnson & Johnson, Employee of Janssen, Ghaith Noaiseh: None declared

Background: RNA sequencing (RNA-seq) has emerged as a widely embraced technique for comprehensive gene expression profiling on a large scale. Nonetheless, there is a current absence of accessible and versatile tools that facilitate efficient exploration of RNA-seq datasets from individuals with Sjogren’s disease (SD) for researchers. Objectives: Our goal is to create user-friendly websites based on R Shiny, designed for gene expression analyses within two distinct SD cohorts: i) an observational disease-control cohort (sicca/Sjogren’s disease), and ii) the TRACTISS randomized clinical trial, comparing Rituximab versus placebo with longitudinal data pre/post-treatment1. Methods: The searchable web interfaces was developed using R Shiny (v.1.7.2) to investigate the associations between individual gene transcript levels and histological as well as clinical parameters, including clinical response. These interfaces incorporate salivary gland (SG) biopsy and peripheral blood (PB) RNAseq data (Table 1) from both a disease-control (Sjogren’s/sicca) cohort and the TRACTISS randomized clinical trial, encompassing longitudinal data from Sjogren’s patients exclusively. Results: The websites enable data exploration and visualization, facilitating the comparison of gene expression levels among defined groups (Sicca/Sjogren or Sjogren: Placebo/Rituximab) based on user-selected clinical variables. Whether the variable is continuous or categorical, users can visualize box plots or correlation plots. Additionally, correlations between individual clinical variables and all genes can be visualized, providing the list of genes significantly correlated with the variable of interest.Differential expression analysis of RNA-seq data is available, offering interactive volcano plots that easily depict the user’s genes of interest. Users can also generate heatmaps using a custom list of genes.For the TRACTISS randomized clinical trial, a time-series analysis of gene expression over time (with three time points for each patient) can be visualized for both SG biopsy and PB RNAseq data. Moreover, gene expression can be stratified based on response criteria, including ESSDAI improvement, CRESS, and STAR composite scores. Conclusion: We have developed two user-friendly websites for Sjogren’s RNA-seq differential expression and time-series analysis. These intuitive platforms offer a diverse selection of interactive plots, enabling researchers to efficiently explore hypotheses, identify expression patterns, and expedite their research with prompt results. All generated results can be downloaded in a high-quality, publication-ready format. REFERENCES: [1] Pontarini E, Sciacca E, Chowdhury F, et al. Serum and tissue biomarkers associated with CRESS and STAR response to B-cell targeted therapy in TRACTISS trial of Sjogren’s syndrome. Arthritis Rheumatol Published Online First: 10 December 2023. doi:10.1002/art.42772 Acknowledgements: NIL. Disclosure of Interests: Elena Pontarini: None declared, Elisabetta Sciacca: None declared, Giulia Cavallaro: None declared, David Galbraith Janssen Pharmaceuticals, Alfredo Pulvirenti: None declared, Ling-Yang Hao Janssen Pharmaceuticals, Kathy Sivils Janssen Pharmaceuticals, Myles Lewis: None declared, Costantino Pitzalis: None declared, Michele Bombardieri Janssen Pharmaceuticals.Figure 1

Background Dysregulated humoral immunity is a hallmark of primary Sjögren's Syndrome (pSS). This dysregulation involves aberrant B-lymphocyte activity resulting in abnormally high immunoglobulin G (IgG) levels and the production of autoantibodies, particularly those reactive with Ro/La ribonuclear complexes. pSS can affect almost any organ system, and current evidence-based therapies only offer some symptom relief, but no licensed therapy has been shown to alter the disease course. Nipocalimab is a high-affinity, fully human monoclonal antibody that reduces circulating IgG levels by selectively blocking the interactions of IgG, including pSS autoantibodies, with the neonatal Fc receptor (FcRn). Nipocalimab has previously induced rapid, safe, and durable serum IgG reductions in healthy volunteers (NCT02828046)[1] and in autoantibody-driven generalized myasthenia gravis (gMG) in adults (Vivacity-MG; NCT03896295)[2], suggesting that nipocalimab may treat a broad range of autoimmune disorders associated with autoantibodies, immune complexes and B-lymphocyte hyperactivity, including pSS. Objectives Here, we describe the key data from Vivacity-MG that illustrate the therapeutic potential of nipocalimab in IgG autoantibody-driven conditions and review the design of a phase 2 study evaluating the efficacy and safety of nipocalimab in patients with pSS (NCT04968912). Methods Study feasibility assessments involved evaluating results from the phase 2 placebo-controlled trial Vivacity-MG. Of the 68 patients enrolled, 54 patients were randomized 1:1:1:1:1 to 4 treatment groups or a placebo group. Results In Vivacity-MG, there were no discontinuations due to treatment-emergent adverse events (TEAEs), severe AEs, or related serious AEs with nipocalimab. The incidence of infections and headaches with nipocalimab were comparable to placebo (Table 1). Treatment with nipocalimab resulted in rapid and dose-dependent reductions in serum total IgG levels and anti-AChR IgG autoantibodies (Figure 1), as compared to placebo. The safety and pharmacodynamic data from Vivacity-MG support the hypothesis that nipocalimab has the potential to treat pSS through lowering pathogenic IgGs. As such, we developed a phase 2, multicenter, randomized, placebo-controlled, double-blind study enrolling adults with moderately-to-severely active pSS. The pSS study consists of a ≤6-week screening period, a 24-week double-blind treatment period, and a 6-week follow-up period. Participants are randomized 1:1:1 to treatment every 2 weeks with intravenous nipocalimab (low or high dose), or placebo, through Week 22. The primary efficacy endpoint is change from baseline in Clinical European League Against Rheumatism Sjögren's Syndrome Disease Activity Index (clinESSDAI) score at Week 24. Safety assessments include TEAEs, abnormal vital signs, and laboratory parameters. Conclusion Vivacity-MG demonstrated that nipocalimab has the potential to offer an important new and targeted treatment option for patients with IgG-mediated diseases. The ongoing phase 2 study evaluates the safety and efficacy of treatment with nipocalimab in patients with moderately-to-severely active pSS. References [1]Ling LE, et al. Clin Pharmacol Ther. 2019;105(4):1031-1039.[2]Ramchandren S, et al. MGFA 2022. Poster 90. Acknowledgements: NIL. Disclosure of Interests Jonathan Hubbard Employee of: Janssen Research & Development, LLC and may own Johnson & Johnson stock or stock options, Kim Campbell Employee of: Janssen Research & Development, LLC and may own Johnson & Johnson stock or stock options, Kathy Sivils Employee of: Janssen Research & Development, LLC and may own Johnson & Johnson stock or stock options, Robert Hoffman Employee of: Janssen Research & Development, LLC and may own Johnson & Johnson stock or stock options, Kim Hung Lo Employee of: Janssen Research & Development, LLC and may own Johnson & Johnson stock or stock options, Jocelyn H Leu Employee of: Janssen Research & Development, LLC and may own Johnson & Johnson stock or stock options, Simon Bowman Consultant of: Abbvie, Astra Zeneca, Galapagos, and Novartis Pharmaceuticals, Sophia Liva Employee of: Janssen Research & Development, LLC and may own Johnson & Johnson stock or stock options, Qing Zuraw Employee of: Janssen Research & Development, LLC and may own Johnson & Johnson stock or stock options, Anne M. Stevens Employee of: Janssen Research & Development, LLC and may own Johnson & Johnson stock or stock options, Leona Ling Employee of: Janssen Research & Development, LLC and may own Johnson & Johnson stock or stock options, Keith Karcher Employee of: Janssen Research & Development, LLC and may own Johnson & Johnson stock or stock options, Sindhu Ramchandren Employee of: Janssen Research & Development, LLC and may own Johnson & Johnson stock or stock options, Hong Sun Employee of: Janssen Research & Development, LLC and may own Johnson & Johnson stock or stock options, R Hal Scofield: None declared, Raphaèle Seror Consultant of: GlaxoSmithKline, Boehringer, Janssen and Novartis; Grant/research support from: GlaxoSmithKline and Amgen, Daniel J. Wallace Consultant of: Amgen, Eli Lilly and Company, EMD Merck Serono, and Pfizer.Table 1TEAE OverviewNipocalimab (n=54)Placebo (n=14)Patients with TEAE, n (%)44 (81.5)11 (78.6)Patients with grade ≥3 TEAE, n (%)04 (28.6)Most frequent TEAEs, n (%)Exacerbation of MG02 (14.3)Headache6 (11.1)1 (7.1)Nasopharyngitis6 (11.1)0Diarrhea6 (11.1)1 (7.1)Patients who discontinued due to TEAEs, n (%)02 (14.3)Patients with serious TEAE, n (%)1 (1.9)*2 (14.3)*Patients with TEAEs deemed related to study drug by investigator, n (%)21 (38.9)1 (7.1)MG, myasthenia gravis; TEAE, treatment-emergent adverse event.*Serious TEAE deemed unrelated to study drug.

Background The presence of circulating rheumatoid factor (RF) and the formation of ectopic lymphoid structures (ELS) in labial salivary glands (SG) of patients with Sjögren’s Syndrome (SS) have been reported as independent risk factors associated with the development of SG B-cell MALT lymphoma (MALT-L). Neoplastic MALT-L B-cells express highly hypermutated B-cell receptors bearing RF immunoreactivity in up to 50% of the cases, but whether their maturation and proliferation is dependent on ELS or is also induced by extrafollicular responses in the SG is currently unclear. The definition of ELS and their association with circulating autoantibodies has so far relied on SG histopathology which bears significant limitations. Conversely, molecular pathology analysis through whole-tissue RNA Sequencing (RNASeq) has allowed a better definition of disease heterogeneity and disease taxonomy. Objectives To perform transcriptomic profiling of SS minor SG tissue characterised by different degrees of inflammatory aggregate organization and to identify transcriptomic clusters and gene signatures associated with peripheral and histological biomarkers of disease. Methods Labial SG were obtained from 99 patients including SS with and without ELS (respectively ELS+ and ELS-, as assessed by immunohistochemistry) and non-specific chronic sialadenitis (Sicca). Total RNA was extracted, complementary DNA libraries were prepared and sequenced. Differentially expressed genes (DEG), deconvolution and pathway analysis were performed. Results Unsupervised gene clustering by differential expression between sicca and SS confirmed a clear transcriptome segregation between the two diagnoses. As expected, in SS SG expression of genes associated with inflammation and adaptive immune responses was upregulated (e.g. CCR7, CD19, CR2, CXCL13, CXCL9, CXCR5, FCRL3, FCRL4, IL21R, MS4A1, PAX5, SLAMF6, TLR10) (Figure A). Bulk RNAseq cell deconvolution confirmed immune cell enrichment (Th, B and plasma cells) in SS SG, especially those ELS+. A three-way comparison among sicca, ELS+ and ELS- SG, showed only a few genes specifically associated to ELS- and sicca, whereas most of the DEGs were either ELS+ specific or common to all SS (Figure B). Results of pathway analysis on ELS+ and SS-associated DEGs showed very similar profiles characterised by adaptive and interferon-associated pathway activation. Weighted Gene Co-Expression Network analysis showed higher activation of anti-viral pathways in ELS- SG, where ELS+ SG were enriched in adaptive immune pathways. Principal component analysis on SS SG mRNA showed that a significant proportion of variability was associated with the presence of RF in the serum of patients independently of the presence of ELS. Surprisingly, transcriptome clustering was closer between ELS+/RF+ and ELS-/RF+ SG than the clustering of ELS+/RF- with ELS-/RF- SG. Accordingly, SG of RF+ patients showed significant upregulation of B cell-associated genes (e.g. CXCL13, MS4A1). However, while SG transcriptomes of ELS+/RF+ were characterised by genes associated with germinal centre formation (e.g IL21, AICDA, LTB), transcriptomes from ELS-/RF+ SG displayed a unique set of DEGs such as BCL2, TRIM22, XAF1, DDX58, DDX60 and IFIT1. Conversely, a similar analysis performed based on anti-Ro/SSA and/or anti-La/SSB did not yield any significant differences in gene expression. Conclusion A comprehensive bulk RNA sequencing analysis of SS and sicca patients SG showed that higher tissue inflammation with features of functional germinal centres is associated to the presence of both RF and ELS, rather than ELS alone. Furthermore, the existence of a RF-driven SG transcriptome independent of the presence of ELS suggest that both follicular and extra-follicular responses support the selection of B-cells with RF immunoreactivity within the SG of SS patients and could be involved in B-cell lymphomagenesis. REFERENCES: NIL. Acknowledgements: NIL. Disclosure of Interests Elena Pontarini: None declared, Davide Lucchesi: None declared, Katriona Goldmann: None declared, Myles Lewis: None declared, Qingxuan Song Employee of: Janssen Pharmaceuticals, Inc., Spring House, PA, USA, Elizabeth Thomas Employee of: Janssen Pharmaceuticals, Inc., Spring House, PA, USA, Ling-Yang Hao Employee of: Janssen Pharmaceuticals, Inc., Spring House, PA, USA, Kathy Sivils Employee of: Janssen Pharmaceuticals, Inc., Spring House, PA, USA, Costantino Pitzalis: None declared, Michele Bombardieri Speakers bureau: UCB, Janssen, Consultant of: GSK, Janssen, MedImmune, Grant/research support from: Janssen, MedImmune.

For many decades, the clinical unmet needs of primary Sjögren's Syndrome (pSS) have been left unresolved due to the rareness of the disease and the complexity of the underlying pathogenic mechanisms, including the pSS-associated lymphomagenesis process. Here, we present the HarmonicSS cloud-computing exemplar which offers beyond the state-of-the-art data analytics services to address the pSS clinical unmet needs, including the development of lymphoma classification models and the identification of biomarkers for lymphomagenesis. The users of the platform have been able to successfully interlink, curate, and harmonize 21 regional, national, and international European cohorts of 7,551 pSS patients with respect to the ethical and legal issues for data sharing. Federated AI algorithms were trained across the harmonized databases, with reduced execution time complexity, yielding robust lymphoma classification models with 85% accuracy, 81.25% sensitivity, 85.4% specificity along with 5 biomarkers for lymphoma development. To our knowledge, this is the first GDPR compliant platform that provides federated AI services to address the pSS clinical unmet needs.

Systemic lupus erythematosus (SLE) is the prototypical multi-system autoimmune disease with diverse clinical features in persons with disease. SLE is also unified by characteristic autoimmunity directed against nucleic acid or nucleoprotein complexes. SLE is both more prevalent and typically exhibits a more severe clinical course in persons with African-American ancestry than in persons with European ancestry. The reasons for this discrepancy remain incompletely understood. GWAS studies of SLE in cohorts of individuals with Amerindian, East Asian and European ancestry have identified > 180 risk loci for SLE across the genome. These loci act in several pathways: clearance of autoantigens, innate immune response to nucleic acids, and lymphocyte activation. Despite these advances in understanding the genetic basis of SLE, a genome-wide association scan (GWAS) of SLE in a cohort of individuals with African-American ancestry has not yet been reported. Here, we report preliminary results of GWAS in 1494 SLE cases and 6076 matched controls with African-American ancestry. Illumina Infinium Omni 1, Omni 1S, Omni 2.5 and OmniExpress platforms were used for genotyping. Genotypes were imputed using the TOPMed reference panel at NHLBI. By defining the contribution common genetic variants to disease risk across the genome, our study represents a step towards understanding the genetic basis of SLE and the increased prevalence and severity of SLE in African ancestry populations. To fully define the relative contribution of environment and genetics to the discrepant SLE severity and prevalence observed in African-American populations, future studies will be necessary. These studies should focus both on comprehensive understanding of the environmental influences and comprehensive assessment of genome- wide genetic variation (i.e. whole-genome sequencing) that impact SLE risk and disease severity. Our results confirm genome-wide significant (P < 5E-8) association with loci ascertained in other populations (STAT4-STAT1, TNIP1, MIR146A, HLA-C4A-C4B, IRF5, BLK, PLAT-IKBKB, RELA-RNASEH2C-OVOL1, ITGAM, and IRF8) and identify several novel genome-wide significant risk loci that are newly described in our study (ENSA, IKBKB/Chr8: Centromere and PCMTD1-ST18). Further, we compared associated variants in our study with those from three large SLE GWAS studies in cohorts of individuals with European, East Asian and Amerindian ancestry. This comparison of SLE risk loci revealed pervasive sharing of SLE genetic risk across ancestral groups. For 70% of the risk loci, the lead marker exhibited nominal association (P < 0.05) with SLE in our GWAS of SLE in African- American persons. Importantly, the association of all such variants cohered with the reported direction in other ancestries. Overall, our findings are consistent with a polygenic contribution to SLE in African-American individuals that is largely shared across populations. We also find association with a rare variant (MAF < 1%) of large effect (OR = 3.91) near a locus previously identified via ImmunoChip (Illumina), PLAT-IKBKB. The lead variant at this locus is non-polymorphic in populations with ancestry outside of Africa. This association explains the increased risk of SLE in ~4% of cases in our cohort. On the one hand, our GWAS of SLE in persons African-American ancestry provides insights that reinforce the conclusions concerning the known risk loci from other ancestries. On the other hand, this mechanism uniquely raises SLE risk in a small proportion of African ancestry individuals with SLE. Together our findings reveal both uniformity and diversity of genetic risk factors impacting SLE development across populations.

Background Sjögren’s disease (SjD) is an autoimmune disease characterized by exocrine gland dysfunction. Long non-coding RNAs (lncRNAs) are a functionally diverse class of non-protein coding RNAs that are longer than 200 nucleotides. Our previous study using whole blood RNA-seq found that lncRNA, LINC01871 , is overexpressed in SjD relative to controls [1]. CRISPR-Cas9 targeting in HSB2 T cells yielded a LINC01871 -/- clone with altered expression of many genes implicated in immune regulation [1]. Objectives The goal of this study was to analyze the gene expression perturbations resulting from the loss of LINC01871 and to characterize the regulation of LINC01871 in both the LINC01871 -/- clone and primary human T cells in response to immune stimuli. Methods Flow cytometry and LegendPlex bead assays were used to compare surface and secreted protein expression changes, respectively, in LINC01871 -/- cells and the parental HSB2 cells. Parental HSB2 T cells, LINC01871 -/- cells, Kasumi-3 myeloid cells, and primary human T cells were stimulated in vitro and changes in gene expression were measured over time using qRT-PCR. Responses to interferons (IFN) were assessed using universal type I IFN (IFNα) or IFNγ. TCR signaling responses were assessed using PMA/Ionomycin (PMA/I) or anti-CD3/CD28 stimulations in the presence or absence of the calcineurin inhibitor, FK506. Results Previous RNA-seq analysis found 1166 differentially expressed (DE) transcripts (log 2 FC ≥1 or ≤-1; p adj ≤0.05) in LINC01871 -/- cells compared to parental HSB2 cells, including many prominent immune regulatory genes. Changes in the basal expression of 7 proteins in LINC01871 -/- cells were confirmed using flow cytometry (significantly decreased: CD8a (p=0.0004), CD30 (p=0.0008), CXCR3 (p=0.037), T-Bet (p=0.0002), and Aiolos (p=0.02); significantly increased: CD226 (p=0.0059) and CD44 (p=0.024)). Analysis of LINC01871 -/- cells revealed a growth inhibition in LINC01871 -/- cells (p=0.0014 at 72h), in which multiple secreted growth and adhesion factors were significantly reduced: GM-CSF (p=2.0e-06), M-CSF (p=2.7e-09), IGBPF4 (p=1.2e-07), s-ICAM1 (p=0.015), MMP9 (p=3.0e-14), and MMP2 (p=6e-08). In contrast, the IL-6 cytokine family member, LIF, was significantly increased in LINC01871 -/- cells (p=1.2e-07). Because HSB2 cells were not responsive to all IFNs, IFN-mediated regulation of LINC01871 expression was examined in the Kasumi-3 myeloid cell line. While LINC01871 expression was not modulated by type I IFN stimulation, it was robustly responsive to IFNγ treatment. Since LINC01871 was expressed in T cells and implicated in T cell pathways, responses to TCR signaling pathways were characterized in HSB2 cells or purified primary human T cells treated with PMA/I. In both cell types, LINC01871 exhibited a prolonged decrease in expression that was abrogated by concurrent treatment with FK506, indicating that LINC01871 is regulated by calcineurin signaling. Treatment of primary human T cells with anti-CD3/CD28 to mimic true TCR engagement resulted in a modest decrease of LINC01871 expression at early time points, followed by an increase in expression with longer stimulation (2d). Conclusion Our findings suggest that LINC01871 is a potential mediator of the dysregulated T cell inflammatory response pathways implicated in SjD pathogenesis. LINC01871 influences the expression of many important immune cell genes and growth factors, is inducible by IFNγ, and is regulated directly by calcineurin signaling and TCR ligand engagement. Although LINC01871 functions are still unknown, observed LINC01871 overexpression in whole blood of SjD cases and after prolonged TCR stimulation in primary human T cells suggests that it could be a biomarker of SjD. References [1]Joachims, et al. Annals of the Rheumatic Diseases 2020;79:90. Disclosure of Interests Michelle L Joachims: None declared, Bhuwan Khatri: None declared, Chuang Li: None declared, Kandice L Tessneer: None declared, John Ice: None declared, Anna M Stolarczyk: None declared, Nicolas Means: None declared, Kiely Grundahl: None declared, Stuart Glenn: None declared, Jennifer Kelly: None declared, David Lewis: None declared, Lida Radfar: None declared, Donald Stone: None declared, Joel Guthridge: None declared, Judith A. James: None declared, R Hal Scofield: None declared, Graham B Wiley: None declared, Jonathan Wren: None declared, Patrick M Gaffney: None declared, Courtney Montgomery: None declared, Kathy Sivils Employee of: Current employee of Janssen., Astrid Rasmussen: None declared, A Darise Farris: None declared, Indra Adrianto: None declared, Christopher Lessard: None declared

Background Sjögren’s disease (SjD) is a chronic, heterogenous autoimmune disorder characterized by inflammatory destruction of the exocrine glands and Ro autoantibodies. Previous studies, most in whole blood, reported transcriptional and cell type differences between SjD subphenotypes. Objectives Evaluate the role of cell type-specific dysregulation in subpopulations of SjD patients having focal lymphocytic sialadenitis and/or anti-Ro/SSA and/or ANA positivity using single-cell RNA-seq (scRNA-seq). Methods Peripherical blood mononuclear cells (PBMCs) stored at -120°C from 30 SjD patients and 10 healthy controls (HCs) were selected from our OMRF Sjögren’s Research Cohort based on ACR/EULAR 2016 criteria. PBMCs were captured, libraries processed using 10X Genomics Chromium v3.1 and sequenced to a depth of ~45,000 reads/cell. 10X Genomics Cell Ranger (v 6.1.2) cell multiplexing pipeline was used to align reads to the GRCh38 human genome and separate combined data to per-sample output files. Seurat R package was used for quality control (QC), integration, clustering, and cell type assignment. Differential expression (DE) analysis was done using Seurat for genes with 10% or more of the cells showing expression. DE transcripts (defined as p adj <0.05) were evaluated using Ingenuity Pathway Analysis (IPA). Results After removing cells with low feature counts, high mitochondrial gene counts, and/or other QC criteria, a total of 331,981 cells were captured. Cells were mapped to 28 clusters using Seurat, then cell types were predicted using marker genes developed from an in-house reference panel of publicly available scRNA-seq datasets. Mapping condensed the 28 observed cell clusters to 19 clusters of specific cell types; 4 clusters remained unidentified. Percentages of each cell type were compared across various subsets of SjD (Ro+; Ro-; Ro-/ANA+; Ro-/ANA-) and HCs. CD14 monocytes were decreased in Ro-/ANA- vs. Ro+ or Ro-/ANA+ cases (p<0.05) and trended downward when compared to HCs (p=0.057). CD8 central memory T cells (TCM) were decreased in Ro+ vs. Ro- or Ro-/ANA- cases (p<0.02) and trended downward when vs. HCs (p=0.076). Conventional dendric cells (DCs) were increased in Ro+ vs. Ro-/ANA- SjD cases (p=0.036) and trended upward in Ro+ vs. Ro- cases (p=0.08) but were not significantly different from HCs. Natural killer (NK) cells were increased in both Ro-/ANA- and Ro- vs. Ro+ (p<0.05). DE analysis between immune cell subsets revealed potential subphenotype-specific differences in pathophysiology. Interferon signaling was upregulated in CD14 monocytes, naïve B, and CD4 central memory (TCM) cells from the Ro+ subset. Ro+ subset also showed upregulation of genes involved in T cell receptor signaling in CD4 cytotoxic T (CTL), CD4 TCM, and CD4 effector memory T (TEM) cells, but downregulation of this pathway in CD8 TCM cells. Ro+ subset also exhibited increased B cell receptor signaling pathway in naïve and memory B cells. Ro+ subset showed downregulation of autophagy in naïve B, CD4 CTL, CD4 TCM, and CD4 TEM cells. Several cell types in Ro+ and Ro-(ANA+/-) subsets showed decreased DE of genes involved in nitric oxide production and NRF2 oxidative stress pathways. Ro- (ANA+/-) vs. HCs showed increased IL6 and TREM1 signaling pathways in CD14 and CD16 monocytes. While Ro+ subsets showed upregulation of B and T cell pathways (shown previously in lupus), Ro- (ANA+/-) showed dysregulation of osteoarthritis and neuroinflammatory pathways. Interestingly, among the DE long intergenic non-coding (linc)RNAs found herein, we observed that LINC01871 , which we previously reported as DE in all subsets of SjD, was overexpressed in CD4 CTL and NK cells in both Ro+ and Ro- (ANA-) vs. HCs. Conclusion This interim analysis shows similarities and differences in the dysregulation of genes and pathways for specific cell subsets from our Ro+ and Ro- (ANA+/-) subsets of SjD, potentially allowing for more tailored diagnostics and interventions for the disease in the future. Disclosure of Interests Chuang Li: None declared, Bhuwan Khatri: None declared, Anna M Stolarczyk: None declared, Kandice L Tessneer: None declared, Astrid Rasmussen: None declared, Joel Guthridge: None declared, Judith A. James: None declared, R Hal Scofield: None declared, He Li Employee of: Current employee of Janssen, A Darise Farris Grant/research support from: Active collaborative research agreement with Janssen, Kathy Sivils Employee of: Current employee of Janssen, Christopher Lessard Grant/research support from: Active collaborative research agreement with Janssen

Background Sjögren’s Disease (SjD) and Systemic Lupus Erythematosus (SLE) are autoimmune diseases with several shared characteristics and similar genome-wide significant associations with the DDX6-CXCR5 locus. DDX6 suppresses interferon-stimulated gene expression and CXCR5 regulates T cell functions implicated in autoimmunity. Objectives To identify and characterize functional SNPs in the DDX6-CXCR5 interval. Methods ImmunoChip data from European populations (3785 SLE cases; 1916 SjD cases; 6893 controls) were imputed and SNP-trait associations tested. Bayesian statistics defined a credible SNP set that was refined using bioinformatic analyses (RegulomeDB, Haploreg, ENCODE, promoter capture Hi-C, eQTLs, etc.). Electrophoretic mobility shift assays (EMSAs) and luciferase expression assays were used to test allele-specific SNP function in EBV-transformed B (EBV B) cells, Daudi B cells, Jurkat T cells, THP1 monocytes, and A253 salivary gland cell lines. Chromatin conformation capture with quantitative PCR (3C-qPCR) was used to assess long-range chromatin interactions. Results Fine mapping of the SjD and SLE associations found similar SNP associations. Bioinformatic analyses identified 5 common SNPs with strong evidence of functionality in immune cell types: rs57494551 in an intron of DDX6, and rs4938572, rs4936443, rs7117261, and rs4938573 in the promoter/enhancer region of DDX6 and CXCR5 . EMSAs and luciferase experiments showed cell type-specific differences in protein binding and promoter or enhancer activity, respectively, at each SNP. Risk allele of rs57494551 increased enhancer activity in B cells and A253 cells (p<0.001), but decreased promoter activity in T cells and A253 cells (p<0.01). SNP rs4938572 is an eQTL of DDX6 in T cells, and the risk allele significantly increased protein binding, promoter and enhancer activity in T cells (p<0.01). Risk allele of rs4938572 also increased promoter activity in A253 cells (p<0.001), but had no effect on promoter or enhancer activity in B cells. SNP rs4936443 showed no promoter or enhancer activity in immune cells, but the risk allele showed significant promoter and enhancer (p<0.001) activity in A253 cells. SNP rs7117261 showed decreased enhancer activity in EBV B cells, T cells, and A253 cells (p<0.05) and increased promoter activity in A253 cells (p<0.001). SNP rs4938573 showed decreased promoter activity in EBV B cells, T cell and A253 cells (p<0.05), decreased promoter activity in EBV B cells (p<0.05), and increased enhancer activity in A253 cells (p<0.0001). Overall, A253 cells exhibited more allele-specific effects on promoter and enhancer activity across the five SNPs compared to tested immune cells. In addition to DDX6 and CXCR5 , rs57494551 and/or rs4938572 are reported eQTLs for several other genes of interest in the local chromatin regulatory network: IL10RA in T cells, TRAPPC4 in salivary gland and activated macrophages, and long non-coding (lnc)RNA AP002954.1 in T cells and whole blood. 3C-qPCR in EBV B and A253 cells showed that the two regulatory regions carrying rs4938572 or rs57494551 interacted with a region upstream of DDX6 that includes AP002954.1 . Hi-C data showed looping between AP002954.1 and the regulatory region carrying rs4938572 and rs57494551 in T cells. Conclusion SjD and SLE share similar genomic architecture across the DDX6-CXCR5 risk interval with several common SNPs showing immune and salivary gland cell type-specific allelic effects on protein binding and/or enhancer/promoter activity. Extensive bioinformatic analyses suggest that the SNPs likely work within the local chromatin regulatory network to regulate cell type-specific expression of several genes on the interval. Ongoing studies will use 3C-qPCR to assess allele-specific chromatin interactions between the SNPs and these genes in different cells types, and CRISPR to determine how the risk alleles alters expression. Disclosure of Interests Mandi M Wiley: None declared, Bhuwan Khatri: None declared, Kandice L Tessneer: None declared, Michelle L Joachims: None declared, Anna M Stolarczyk: None declared, Anna Nagel: None declared, Astrid Rasmussen: None declared, Simon J. Bowman Consultant of: Abbvie, Galapagos, and Novartis in 2020-2021, Lida Radfar: None declared, Roald Omdal: None declared, Marie Wahren-Herlenius: None declared, Blake M Warner: None declared, Torsten Witte: None declared, Roland Jonsson: None declared, Maureen Rischmueller: None declared, Patrick M Gaffney: None declared, Judith A. James: None declared, Lars Ronnblom: None declared, R Hal Scofield: None declared, Xavier Mariette: None declared, Wan Fai Ng: None declared, Kathy Sivils Employee of: current employee of Janssen., Gunnel Nordmark: None declared, Betty Tsao: None declared, Christopher Lessard: None declared