A1.15 IL-6 driven Stat3 phosphorylation in circulating lymphocytes is specific for CD4<sup>+</sup>T cells in early rheumatoid arthritis
Authors: Amy E Anderson, Arthur G Pratt, Ben Hargreaves, Christine Routledge, John Paul Doran, Philip Mawson, John D Isaacs
Journal: Annals of the Rheumatic Diseases (2014)

<h3>Background</h3> To expedite the diagnosis of rheumatoid arthritis (RA), and facilitate individualised treatment selection for patients with this heterogeneous disease, an improved understanding of cell signalling pathways variously deregulated at its earliest stages will be required. We recently identified a transcriptional signature in circulating CD4<sup>+</sup> T-cells, which predicted RA progression amongst patients attending an early arthritis clinic. The signature contained an over-representation of Stat3 regulated genes, whose expression in turn correlated with serum IL-6. We therefore sought an improved understanding of Stat signalling amongst immune cell subsets of an independent early arthritis patient cohort. <h3>Methods</h3> Newly presenting patients naïve to immunomodulatory treatment (including steroids) were recruited from an early arthritis clinic, and followed until diagnoses were confirmed. Basal and IL-6-induced expression of intracellular phospho-Stat3 (pStat3) / pStat1 was determined in T-cell and B-cell subsets by whole blood flow cytometric analysis, which included assessment of surface IL-6R expression. Contemporaneous serum levels of IL-6 and other STAT3 utilising factors, along with those of IL-6R and gp130, were measured by immuno-assay. <h3>Results</h3> Analysis, currently complete for 94 early arthritis patients (described here), will be presented for 208 recruited in total. Basal pStat3 levels appear highest in circulating CD4<sup>+</sup> T-cells. Basal pStat3 expression correlates with serum IL-6 levels uniquely in CD4<sup>+</sup> T-cells compared with CD8<sup>+</sup> T-cells and B-cells – and most prominently in the naïve subset. The expected pStat3 induction following IL-6 stimulation, observed in all subsets, was also most pronounced in CD4<sup>+</sup> T-cells. These observations mirror the pattern of surface IL-6R expression, this being highest on CD4<sup>+</sup> T-cells (and in particular naïve and central memory subsets). When patients are categorised by diagnostic outcome, ACPA-negative RA patients have higher basal CD4<sup>+</sup> T-cell pStat3 expression than ACPA-positive RA, inflammatory non-RA and non-inflammatory arthritis patients – a pattern not seen in CD8<sup>+</sup> T-cells or B-cells which corroborates our previous observations in respect of Stat3 target gene expression. <h3>Conclusions</h3> Our findings support a particular role for IL-6-driven CD4<sup>+</sup> T cell activation, primarily via Stat3, during the induction of RA. Given the pattern of surface IL-6R expression, a critical role in this setting for <i>classical</i> IL-6 signalling is suggested (as opposed to <i>trans</i> signalling, which is not dependent on IL-6R surface expression). Expression of pStat3 in CD4<sup>+</sup> T-cells may serve as a biomarker for predicting the evolution of RA in ACPA-negative patients and may also be a useful tool for predicting efficacy of therapies which target IL-6 signalling.