P093 A CD25 enhancer variant linked to juvenile idiopathic arthritis skews CD4+ T-Cell responses towards a proinflammatory profile, modifiable by low-dose IL-2

Abstract Background/Aims <jats:p/>Juvenile idiopathic arthritis (JIA) affects approximately 12,000 children in the UK, half of whom fail to improve on treatment. There is therefore a significant clinical need for new therapies in JIA. A CD25 (IL2RA) enhancer variant associated with JIA has been shown to affect CD25 expression on T-cell subsets (expression quantitative trait locus or eQTL). More recently using mice, researchers have shown this variant skews the balance of T-cell homeostasis. The aim of this study was to translate these findings in humans. Methods <jats:p/>Whole blood from healthy adult volunteers who were major (n = 6) or minor (n = 6) allele homozygotes for the IL2RA enhancer variant, rs12722495, were obtained through the NIHR bioresource center. Flow cytometry was used to observe Tregs (CD25+FOXP3+)/CD4+ populations at steady-state and CD25 expression on naïve CD4+ T-cells following stimulation using anti-CD3 (5µg/mL) and CD28 (2.5µg/mL) for 24, 48 and 72-hours. Naïve CD4+ T-cells were polarized towards Treg or Th17 cell-states over 7-days and identified by flow cytometry. For some conditions, low-dose IL-2 (50U) or anti-IL-2 (10µg/mL) were added to the media. Efficacy of induced Tregs (iTregs) were determined by suppression assay and supernatants from differentiations were analyzed for IL-10 (iTregs) and IL-17A (Th17s) by ELISA. Results <jats:p/>No differences in Treg populations or CD4+ T-cell naivety (CD45RA+) were found between genotype groups at steady-state. In naïve CD4+ T-cells, following 48-hour stimulation, significantly fewer CD25+ T-cells were observed in the minor allele group, with a similar trend observed after 72 hours. Although not significant, the same trends were observed with CD25 mean fluorescent intensity. CD69 expression was similar between groups, suggesting no general stimulation impairment. Addition of IL-2 promoted the upregulation of CD25 so no difference was observed. We next assessed how impairment in IL2RA induction skews polarization of naïve CD4+ T-cells. Following Treg differentiation, similar proportions of iTregs were observed between genotype groups. IL-2 treatment significantly increased percentages of iTregs in the major allele genotype, suggesting these cells have heightened sensitivity to IL-2. iTregs from both groups had similar suppressive capacities. Following Th17 differentiation, a trend was observed for more Th17 cells within the minor allele group. As expected, with addition of IL-2, fewer IL-17A+ cells were observed within both groups. In addition, we characterized these cells by looking at cytokine secretion. Interestingly, iTregs and Th17s from the major allele group produced significantly less IL-10 and more IL-17A, respectively. Conclusion <jats:p/>We replicated the CD25 eQTL and observed a delayed response in individuals carrying the minor allele as seen in mice. This impairment, although not significant, disrupts IL-2 signaling, shifting polarization towards a pro-inflammatory state. Furthermore, we show that IL-2 compensates for the effect of the variant, identifying a subset of patients who may benefit from low-dose IL-2 therapy.