Time-resolved immune dynamics in rheumatoid arthritis under methotrexate therapy
Time-resolved immune dynamics in rheumatoid arthritis under methotrexate therapy
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. 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. 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). 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 3â€™ Reagent Kit (v3/v3.1).