054. The Importance of IL-6-STAT3 Mediated Activation of Circulating CD4⁺ T Cells in the Pathogenesis of Early Seronegative Rheumatoid Arthritis: A Validation Study

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Background: Early diagnosis of RA improves outcomes but is challenging, particularly among ACPA-negative individuals. Previously we identified an IL-6-mediated CD4⁺ T cell transcriptional signature, enriched for signal transduction and activation of transcription-3 (STAT3) target genes, which had discriminatory value for this purpose. In the present work we sought independent replication of those findings, the development of a more readily applicable diagnostic assay and insight into mechanisms of disease induction.

Methods: Among 210 early arthritis patients attending the Newcastle Early Arthritis Clinic (NEAC) who were naive to immunomodulatory treatment, serum cytokine levels were ascertained by immuno-assay. Constitutive and IL-6-induced expression of phosphorylated STAT1 and 3 (pSTAT1/3) were determined in paired fresh circulating lymphocytes using flow cytometry. Finally, contemporaneous CD4⁺ T cell gene expression was measured in highly purified, fresh CD4⁺ T cells by hybridizing high-integrity RNA isolates to Illumina Human HT12 BeadChips, and employing appropriate microarray normalization algorithms. Patients were followed up for >12 months and diagnostic outcomes confirmed. Analyses included non-parametric ANOVA (Dunn’s post-hoc group comparisons), Spearman’s rank correlation, multiple regression and hierarchical clustering.