biostudies-arrayexpressAnela TosevskaHomo sapienshttps://www.ebi.ac.uk/biostudies/studies/E-MTAB-17153Methotrexate (MTX) serves as the primary treatment for rheumatoid arthritis (RA), though its mechanism of action is not yet fully understood. The goal of this study was to map the cellular and molecular changes triggered by MTX across different points in time. Using a prospective longitudinal design, newly diagnosed RA patients starting MTX therapy were closely monitored over the course of treatment. Single-cell RNA sequencing (scRNA-seq) of peripheral blood mononuclear cells (PBMCs) was performed on 5 patients at baseline, at successive time points during therapy, and in healthy controls. The findings revealed that MTX drives swift immune system restructuring — changes that emerged as early as three weeks into treatment and appeared before any measurable clinical benefit.biostudies-arrayexpressGrowth Protocol - Not applicable. This study profiles primary human immune cells isolated directly from patient peripheral blood which were not subjected to ex vivo expansion or culturing prior to downstream assays.Library Construction - Longitudinal PBMC samples (baseline, week 3, week 6, and week 12) from a single RA patient were first multiplexed by incubation with distinct DNA-labeled antibodies (BioLegend TotalSeq-A) before pooling and processing as a single multiplexed sample for simultaneous single-cell RNA-seq and antibody-derived tag (ADT) library construction using the 10x Genomics Next GEM Single Cell 3’ Reagent Kit (v3/v3.1).Sample Collection - Peripheral blood was collected from rheumatoid arthritis (RA) patients and age-, sex-, and ethnicity-matched healthy controls via venipuncture. For RA patients, blood samples were collected longitudinally over a 12-week period at specific clinical intervals (including baseline, 3 weeks, 6 weeks and 12 weeks post-treatment initiation). Peripheral blood mononuclear cells (PBMCs) were immediately isolated from whole blood using density gradient centrifugation (e.g., Ficoll-Paque), washed, and cryopreserved in liquid nitrogen until further processing to minimize batch effects.Sample Treatment - Healthy control samples received no therapeutic interventions. For the longitudinal patient cohorts, individuals were treated clinically with standard first-line Methotrexate (MTX) therapy. PBMCs collected at subsequent time points (Weeks 3, 6 and 12) represent the in vivo effects of continuous MTX exposure on peripheral immune cell dynamics relative to the untreated baseline (Week 0).Sequencing - The libraries were sequenced using the Illumina HiSeq 3000/4000 platform.Library Construction - Single-cell RNA-seq libraries were constructed from individual healthy control PBMC samples using the Chromium Controller and the 10x Genomics Next GEM Single Cell 3’ Reagent Kit (v3/v3.1) via cDNA fragmentation, adapter ligation, and sample index PCR amplification according to the manufacturer’s instructions.OrganizationMINSEQE ScoreAssays and DataProcessed DataMAGE-TAB FilesSequence Alignment - Raw sequencing data underwent pre-processing and alignment to the GRCh38 human reference genome as well as demultiplexing of the antibody-linked barcodes using Cell Ranger (v6.1.2, 10x Genomics) generating count data.Data Transformation - Seurat v5.0.01 package was used for data normalization (using log normalization with a scale factor of 10000) and scaling.MetabolomicsUnknownTranscriptomicsGenomicsProteomicsIllumina HiSeq 3000Rheumatoid arthritis (RA) is characterized by immune dysregulation, including alterations in peripheral blood mononuclear cell (PBMC) populations and aberrant cytokine signaling. Methotrexate (MTX) is the preferred first-line treatment for RA, yet its precise mechanisms of action remain incompletely understood. This study employed a multi-omics strategy—combining single-cell RNA sequencing (scRNA-seq) and immunophenotyping—to identify key effector peripheral immune cells and their cellular responses in RA patients over 12 weeks of MTX treatment. In our study, MTX was associated with significant immune modulation, including the restoration of naïve T and B cells and reductions in T cell memory subsets with these effects detectable as early as three weeks post-treatment. Plasmablast levels also emerged as a potential biomarker for early therapeutic response, reflecting MTX’s impact on immune homeostasis. Transcriptional analysis revealed modulation of key pathways, including TNF-α signaling, B cell receptor signaling, and T cell receptor-mediated apoptosis. Network analysis identified critical regulatory hubs, such as EGR1, JAK2, and SOCS1, in monocytes and CD4 memory T cells, highlighting these cell types as key mediators of MTX’s effects. In conclusion, these findings advance our understanding of MTX’s effects on immune cell dynamics at different stages of treatment, showing for the first time the early cellular changes leading to immune modulation in RA. Altogether, our results provide the foundation for further mechanistic investigations into MTX.RNA-seq of coding RNA from single cellsHomo sapiensTime-resolved immune dynamics in rheumatoid arthritis under methotrexate therapyAnna MrakTeresa Preglej,Anela Tosevska,Marie Brinkmann,Philipp Schatzlmaier,Elisabeth Simader,Daniela Sieghart, Philipp Hofer,Thomas Krausgruber,Lina Dobnikar,Christoph Bock,Thomas Karonitsch,Renate Kain,Hannes Stockinger,Wilfried Ellmeier,Daniel Aletaha, Lisa Göschl,Michael BonelliAnela TosevskafalseTime-resolved immune dynamics in rheumatoid arthritis under methotrexate therapyMethotrexate (MTX) serves as the primary treatment for rheumatoid arthritis (RA), though its mechanism of action is not yet fully understood. The goal of this study was to map the cellular and molecular changes triggered by MTX across different points in time. Using a prospective longitudinal design, newly diagnosed RA patients starting MTX therapy were closely monitored over the course of treatment. Single-cell RNA sequencing (scRNA-seq) of peripheral blood mononuclear cells (PBMCs) was performed on 5 patients at baseline, at successive time points during therapy, and in healthy controls. The findings revealed that MTX drives swift immune system restructuring — changes that emerged as early as three weeks into treatment and appeared before any measurable clinical benefit.2026-07-01T00:00:00Z2026-07-01T12:27:12.782Z2026-06-12T11:13:56.421ZE-MTAB-17153ERP194979EFO_0004170EFO_0003789EFO_0005684EFO_0004917EFO_0005518EFO_0003816EFO_0003969EFO_000418410.1101/2025.01.14.629357