Background: The pathogenesis of rheumatoid arthritis (RA) is complex and incompletely understood. B-cells are significant contributors to RA pathogenesis through autoantibody production and cytokine secretion. Understanding gene expression profiles in B-cells can elucidate immunological mechanisms at different stages of the disease and identify biomarkers of disease activity. Of particular importance are biomarkers indicating response to therapeutic interventions such as Methotrexate (MTX), a cornerstone in RA treatment.

Objectives: This study aims to identify gene expression profiles of CD19+ B-cells from RA patients at different clinical disease stages, including before and after initiation of MTX treatment, for better understanding of disease pathogenesis and identification of putative biomarkers.

Methods: Blood samples (n = 61) were collected from two RA cohorts; newly diagnosed RA patients from the NOR-VEAC cohort at diagnosis (n = 10) and 3 months following MTX initiation (n = 9), and patients with established RA on MTX treatment in remission from the ARCTIC REWIND cohort at inclusion (n = 18) and at 8 or 12 months follow-up (n = 15), as well as from healthy controls from the Norwegian bone marrow registry (n = 9). In both patient cohorts, clinical data, including DAS28, were collected. CD19+ B-cells were isolated from the blood using magnetic beads with the EasySep Human CD19 Positive Selection Kit from STEMCELL Technologies. Total RNA was extracted and ribosomal RNA depleted prior to sequencing on the Illumina NovaSeq6000 platform, resulting in a total of 5.4 billion reads (mean of 89 million reads per sample). Statistical differential expression analysis was performed using the limma package in R.

Results: Patients were matched across the cohorts regarding age, gender, and smoking history (Table 1). The newly diagnosed patients showed high disease activity measured with DAS28, while the MTX-treated patients with established RA were in remission. Preliminary results showed few gene expression differences between CD19+ B-cells from newly diagnosed patients at baseline compared to healthy controls. However, 3 months following MTX initiation, the newly diagnosed patients displayed expression changes in their CD19+ B-cells for about 300 genes compared to baseline. In patients with established RA in remission on MTX treatment, we observed a large number of significantly differentially expressed genes both compared to healthy controls, as well as intra-individually over time (samples from 8 or 12 months versus samples from inclusion). There was substantial overlap in the significant genes observed in all analyses. Among the identified genes, several harbor polymorphisms previously reported to be RA associations through genome-wide association studies. These findings align with a previous study on the same patient populations (Heinicke, 2021), where we found differentially expressed microRNAs in CD19+ B cells in RA patients and predicted their putative target genes, known to be involved in B-cell activation, differentiation, and B-cell receptor signaling. Interestingly, several of these genes, predicted to be regulated by RA-associated miRNAs, were observed to be differential expressed in the current study. Next, we further will perform pathway and gene enrichment analyses to explore the molecular mechanisms and regulatory networks potentially altered at the different disease stages. In line with this, we will investigate the gene expression profiles in-depth in a B-cell single-cell dataset generated from a subset of these patients to enhance our understanding of which CD19+ B-cell subsets may contribute to these processes.

Conclusion: Few transcriptional differences were observed in B-cells between newly diagnosed, untreated patients and healthy controls, while MTX-treated patients showed substantial gene expression differences, likely accumulating over time, despite disease remission. This suggests distinct B-cell transcriptional changes associated primarily with MTX treatment, not with disease onset or progression. Our results offer potential insights into disease monitoring and novel biomarker identification.

REFERENCES: [1] Heinicke F, Zhong X, Flåm ST, et al. MicroRNA Expression Differences in Blood-Derived CD19+ B Cells of Methotrexate Treated Rheumatoid Arthritis Patients. Front Immunol . 2021;12:663736. Published 2021 Apr 9. doi:10.3389/fimmu.2021.663736.

Acknowledgements: NIL.

Disclosure of Interests: Maria Dehli Vigeland: None declared , Fatima Heinicke: None declared , Siri Tennebø Flåm: None declared , Siri Lillegraven: None declared , Maria Mjaavatten: None declared , Silje Syversen: None declared , Espen Haavardsholm Pfizer, Eli Lilly, Novartis and AbbVie, Pfizer, Eli Lilly, Novartis and AbbVie, Benedicte A. Lie: None declared .

© The Authors 2025. This abstract is an open access article published in Annals of Rheumatic Diseases under the CC BY-NC-ND license ( http://creativecommons.org/licenses/by-nc-nd/4.0/ ). Neither EULAR nor the publisher make any representation as to the accuracy of the content. The authors are solely responsible for the content in their abstract including accuracy of the facts, statements, results, conclusion, citing resources etc.