We present a unique analysis of the CD4 T-cell transcriptome in a well-characterised inception cohort of early arthritis patients attending a routine EAC in UK. As a potential diagnostic tool, it is significant that our 12-gene ‘RA expression signature’ () performed best among the diagnostically challenging ACPA-negative UA patient group. Intriguingly, these findings support the involvement of CD4 T cells in both ACPA positive and negative disease.
The signature's sensitivity and specificity (0.85 and 0.75) for predicting subsequent RA in seronegative UA patients equate to a positive likelihood ratio of 3.4, indicating that a prior probability of 25% for RA progression among this cohort (13 of the 49 patients progressed to RA) doubles to 53% for an individual who has been assigned a positive SVM classification.35
Moreover, of the 13 ACPA-negative UA patients who progressed to RA in our cohort, 8 fell into an ‘intermediate’ risk category for RA progression according to the validated Leiden prediction score.5
Encouragingly, all but one of these patients were correctly classified based on their 12-gene expression profile. Our proposal that this approach might add value to existing algorithms for the diagnosis of ACPA-negative UA is further supported by the construction of ROC curves comparing the Leiden prediction rule with a modified risk metric that incorporates features of our gene signature ().
Our data indicate that PB CD4 T cells in early RA are characterised by a predominant upregulation of biological pathways involved in cell cycle progression (ACPA-positive) and survival, death and apoptosis (ACPA-negative) (; also online supplementary gene lists 5–6). Pathway analysis also suggested that T-cell development and differentiation were deregulated in both RA serotypes (online supplementary gene list 7). These findings concur with previous observations of impaired T-cell homeostasis in RA, characterised by increased turnover, telomere shortening and immunosenescence.36
Given the well-characterised importance of the STAT3 signalling pathway in both oncogenesis and T-cell survival, it was notable that five genes from our statistically robust 12-gene RA signature are downstream of STAT3 signalling.20–25
The degree to which these genes sub-cluster according to the expression pattern among individuals in both the training and validation cohorts () presumably reflects their co-regulation by STAT3. Their upregulation was generally most pronounced in ACPA-negative RA (; also online supplementary figure S4A–C), explaining why the predictive utility of the 12-gene signature was optimal in this disease subset.
Our observation that increased serum IL-6 levels among EAC attendees may predict a diagnosis of RA versus alternative arthritides is consistent with findings of previous biomarker studies,38
but ours is the first demonstration of a particular association with ACPA-negative disease (). Striking correlations were seen between PB CD4 T-cell expression of several STAT3-inducible genes and paired, contemporaneous serum IL-6 concentrations, which were independent of alternative acute phase markers (; also online supplementary figures S5A–D and table S6). STAT3 phosphorylation and downstream transcription is initiated by ligation of the cell-surface gp130 co-receptor by a range of ligands, including IL-6.40
We measured IL-6 in particular because of its recognised role as a pro-inflammatory cytokine in RA,41
and we excluded similar relationships with sIL6R (a surrogate of IL-6R trans-signalling) and other relevant substrates of STAT3 signalling. Therefore, the STAT3-inducible gene expression signature that we have identified does appear to be downstream of IL-6 signalling. The capacity of IL-6 alone to induce the STAT-3-regulated elements of our early RA gene expression signature in primary CD4 T cells was confirmed in vitro (online supplementary figures S6 and S7).
In conclusion, our data provide strong evidence for the induction of an IL-6-mediated STAT3 transcription programme in PB CD4 T cells of early RA patients, which is most prominent in ACPA-negative individuals and which contributes to a gene expression ‘signature’ that may have diagnostic utility. Furthermore, our findings could pave the way for a novel treatment paradigm, whereby emerging drugs targeting the IL-6-gp130-STAT3 ‘axis’42
find a rational niche as first choice agents in the management of ACPA-negative RA. Studies, such as ours, should ultimately contribute to the realisation of true ‘personalised medicine’ in early inflammatory arthritis, in which complex heterogeneity is stratified into pathophysiologically and therapeutically relevant subsets, with clear benefits in terms of clinical outcome and cost.