biostudies-arrayexpressElena PontariniHomo sapienshttps://www.ebi.ac.uk/biostudies/studies/E-MTAB-15225Sjogren's disease patients received Rituximab (anti-CD20 depleting monoclonal antibody) or placebo treatment. This study aims to identify the transcriptomic features of salivary gland biopsies, the target tissue of the autoimmune response in Sjögren’s disease. It examines transcriptomic changes in salivary gland tissue from patients treated with Rituximab compared to those in the placebo group, alongside longitudinal changes pre/post treatment.biostudies-arrayexpressLibrary Construction - RNA sequencing libraries were prepared using the NEBNext Ultra RNA Library Prep Kit for Illumina. Briefly, mRNA was first enriched with Oligod(T) beads and fragmented for 15 minutes at 94°C. First-strand and second-strand cDNA were subsequently synthesized. cDNA fragments were end-repaired and adenylated at 3’ends, and universal adapters were ligated to cDNA fragments, followed by index addition and library enrichment by PCR with limited cycles. The sequencing library was validated on TapeStation and quantified with Qubit 2.0 Fluorometer and quantitative PCR (KAPA Biosystems).Growth Protocol - NASample Collection - One LSG lobule per patient was stored in RNAlater solution for 24 hours at 4C and transferred at −80C for long-term storage.Nucleic Acid Extraction - SG RNA was extracted usingRNeasy Micro Kit (Qiagen) as per the manufacturer’s instructions, quantified and checked for integrity using Qubit-2.0 Fluorometer(Invitrogen) and TapeStation (Agilent Technologies), respectively.Sample Treatment - Patients received either two doses, two weeks apart, of intravenous rituximab (1,000 mg) or placebo (250 mL saline) in two courses at weeks 0 and 2, 24, and 26 in combination with corticosteroid in both arms.Sequencing - Libraries were sequenced on Illumina HiSeq 4000, using 2×150bp paired end configuration, 50 million reads/sample. One mismatch was allowed for index sequence identification.OrganizationMINSEQE ScoreAssays and DataMAGE-TAB FilesData Transformation - Variance stabilising transform (VST) normalised gene expression levels were used.Sequence Alignment - NAUnknownTranscriptomicsGenomicsProteomicsIllumina HiSeq 4000<h4>Objective</h4>This study aimed to identify peripheral and salivary gland (SG) biomarkers of response/resistance to B cell depletion based on the novel concise Composite of Relevant Endpoints for Sjögren Syndrome (cCRESS) and candidate Sjögren Tool for Assessing Response (STAR) composite endpoints.<h4>Methods</h4>Longitudinal analysis of peripheral blood and SG biopsies was performed pre- and post-treatment from the Trial of Anti-B Cell Therapy in Patients With Primary Sjögren Syndrome (TRACTISS) combining flow cytometry immunophenotyping, serum cytokines, and SG bulk RNA sequencing.<h4>Results</h4>Rituximab treatment prevented the worsening of SG inflammation observed in the placebo arm, by inhibiting the accumulation of class-switched memory B cells within the SG. Furthermore, rituximab significantly down-regulated genes involved in immune-cell recruitment, lymphoid organization alongside antigen presentation, and T cell co-stimulatory pathways. In the peripheral compartment, rituximab down-regulated immunoglobulins  and auto-antibodies together with pro-inflammatory cytokines and chemokines. Interestingly, patients classified as responders  according to STAR displayed significantly higher baseline levels of C-X-C motif chemokine ligand-13 (CXCL13), interleukin (IL)-22, IL-17A, IL-17F, and tumor necrosis factor-α (TNF-α), whereas a longitudinal analysis of serum T cell-related cytokines showed a selective reduction in both STAR and cCRESS responder patients. Conversely, cCRESS response was better associated with biomarkers of SG immunopathology, with cCRESS-responders showing a significant decrease in SG B cell infiltration and reduced expression of transcriptional gene modules related to T cell costimulation, complement activation, and Fcγ-receptor engagement. Finally, cCRESS and STAR response were associated with a significant improvement in SG exocrine function linked to transcriptional evidence of SG epithelial and metabolic restoration.<h4>Conclusion</h4>Rituximab modulates both peripheral and SG inflammation, preventing the deterioration of exocrine function with functional and metabolic restoration of the glandular epithelium. Response assessed by newly developed cCRESS and STAR criteria was associated with differential modulation of peripheral and SG biomarkers, emerging as novel tools for patient stratification.RNA-seq of coding RNAHomo sapiensLactate signalling leads to aggregation of immune-inflammatory hotspots and SLC5A12 blockade promotes their resolutionSerum and Tissue Biomarkers Associated With Composite of Relevant Endpoints for Sjögren Syndrome (CRESS) and Sjögren Tool for Assessing Response (STAR) to B Cell-Targeted Therapy in the Trial of Anti-B Cell Therapy in Patients With Primary Sjögren Syndrome (TRACTISS)Michelangelo Certo, Elena Pontarini, Sebastian G Gilbert, Ronny Schmidt, Jason D Turner, Davide Lucchesi, Daria Apostolo, Giulia Cavallaro, Charlotte G Smith, Serena Colafrancesco, Joana Campos, Saba Nayar, Christoph Schröder, Benjamin A Fisher, Fabian Spill, Michele Bombardieri, Claudio MauroPontarini E, Sciacca E, Chowdhury F, Grigoriadou S, Rivellese F, Murray-Brown WJ, Lucchesi D, Fossati-Jimack L, Nerviani A, Jaworska E, Ghirardi GM, Giacomassi C, Emery P, Ng WF, Sutcliffe N, Everett C, Fernandez C, Tappuni A, Seror R, Mariette X, Porcher R, Cavallaro G, Pulvirenti A, Verstappen GM, de Wolff L, Arends S, Bootsma H, Lewis MJ, Pitzalis C, Bowman SJ, Bombardieri M;Elena PontarinifalseRNA-sequencing from human salivary glands from Sjogren's disease patients recruited in the TRACTISS randomized clinical trialSjogren's disease patients received Rituximab (anti-CD20 depleting monoclonal antibody) or placebo treatment. This study aims to identify the transcriptomic features of salivary gland biopsies, the target tissue of the autoimmune response in Sjögren’s disease. It examines transcriptomic changes in salivary gland tissue from patients treated with Rituximab compared to those in the placebo group, alongside longitudinal changes pre/post treatment.2025-06-30T00:00:00Z2025-06-13T15:48:52.496Z2025-06-13T15:48:52.496ZE-MTAB-15225ERP173460EFO_0002944EFO_0004170EFO_0003789EFO_0004917EFO_0005518EFO_0003816EFO_0003738EFO_0004184EFO_000396910.1002/art.42772