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Objectives Antimalarials (AM) and glucocorticoids (GC) are commonly used for treating autoimmune rheumatic diseases, but their molecular effects and potential synergistic interactions remain unclear. This study aimed to address this gap by investigating transcriptome-based molecular pathways associated with AM and GC use, and with their interactionsMethods We analyzed bulk RNA sequencing data from 582 whole-blood samples using a cross-sectional cohort from the European PRECISESADS project. The patients were diagnosed with systemic lupus erythematosus (SLE), primary Sjögren’s disease (pSjD), undifferentiated connective tissue disease (UCTD), or mixed connective tissue disease (MCTD), and were grouped into four categories based on current treatment: no current exposure to AM, GC, or any immunosuppressants (off-treatment; n = 244), AM monotherapy (n = 198), GC monotherapy (n = 45), or AM and GC combined therapy but no immunosuppressants (n = 95) ( table 1). We performed differential gene expression (DGE) analysis comparing each treatment group (AM, GC, and AM+GC) against the off-treatment group as reference, followed by gene set enrichment analysis (GSEA). Overrepresentation analysis (ORA) was conducted on genes exhibiting amplified effect sizes under combination treatment. The identified signatures were validated using a longitudinal independent cohort of SLE patients from the Johns Hopkins Lupus Center (JHLC), while their associated cell types were validated using publicly available scRNA-seq data from peripheral blood mononuclear cells (PBMCs) of SLE patients (GSE135779). Additionally, flow cytometry data from 541 PRECISESADS whole-blood samples were analyzed using defined treatment groups as a regressor in a beta-regression model.Results Combination treatment with AM and GC resulted in a higher number of differentially expressed genes and larger effect sizes compared to AM or GC monotherapy. Functional analyses identified 78 shared immune-related pathways (ORA adj. p-value < 0.05, GSEA adj. p-value < 0.05). Notably, two pathways were validated in the independent JHLC cohort, both associated with B cell activation. Consistent with these transcriptional changes, B cell proportions were reduced under combination treatment in the PRECISESADS cohort.Abstract PO:08:216 Table 1Sample numbers per group per cohortAbstract PO:08:216 Figure 1Number of differentially expressed genes between treatment regimens and off-treatmentConclusions Our findings suggest potential synergistic effects between AM and GC when administered in combination, evidenced by increased numbers of differentially expressed genes, with enhanced modulation of key immune processes, particularly B cell activation pathways.