AB0027 INCREASED CIRCULATING CD39+FOXP3+CD4+ TREG CELLS IN EARLY RHEUMATOID ARTHRITIS FACILITATE THE ANTIINFLAMMATORY ACTION OF METHOTREXATE

Abstract

Background:Methotrexate (MTX) remains the first line of treatment in Rheumatoid Arthritis (RA)1,2. Inhibition of AICAR transformylase by MTX results in augmented release of adenine nucleotides to the extracellular space1; these are rapidly hydrolysed by the combined action of ectonucleotidases CD39 and CD79 rendering the antiinflammatory agent adenosine1. CD39, the rate-limiting enzyme in this cascade, is highly expressed by a subset of human FoxP3+CD4+ regulatory T cells (Treg39+)2-4 and MTX may act synergistically with Tregs in the control of inflammation.Objectives:To study the expression of CD39 on circulating Treg cells of untreated early Rheumatoid Arthritis (ERA) patients and its relation with the ex vivo effect of MTX.Methods:Peripheral blood was drawn from 22 DMARD- and steroid- naïve ERA patients with a disease duration < 24 weeks, 15 longstanding RA patients (LRA, disease duration > 2 years) and 37 age and gender-matched healthy controls (HC). LRA patients were receiving low-dose weekly MTX and were naïve for biologicals. 10 ERA patients who had achieved remission 12 months after initiating MTX donated blood for a second time (ERA-R). The frequency of Treg and Treg cell subsets was assessed by flow cytometry. CD4+CD25+CD127- (total T reg), CD4+CD27+CD127-CD39+ Treg (Treg39+) and CD4+CD25-CD39- responder T (Tresp39-) cells were isolated by Ficoll-Hypaque, followed by sorting. The suppressor potency of Tregs was assessed in cocultures of isolated Tregs with Tresp, established at different Treg/Tresp ratios. Proliferation was determined by CFSE dilution; cytokine secretion was measured by ELISA of culture supernatants.Results:As previously described5, ERA but not LRA patients demonstrated a superior frequency of circulating Treg (CD4+CD25+CD127-FoxP3+) cells. In addition, the proportion of Tregs that expressed CD39 (Treg39+) was significantly increased in ERA but not LRA. Total ERA Tregs were significantly more potent suppressors of proliferation, TNFα and IFNγ secretion when compared with HC or LRA Tregs, and this difference was partially and significantly abrogated in the presence of adenosine deaminase, or the adenosine A2A receptor (A2AR) antagonists DMPX (3,7-dimethyl-1-propargylxanthine) or ZM 241385, but not in the presence of the adenosine A1 receptor antagonist DPCPX (8-cyclopentyl-dipropylxanthine). When MTX was added to the culture medium, the suppressor potency of total Tregs was further enhanced in all 3 groups of patients, and this enhancement was significantly higher in ERA total Treg/Tresp39- cocultures as compared with HC or LRA total Treg/Tresp39- cocultures. The effect of MTX was also partially and significantly abrogated by adenosine deaminase, DMPX or ZM 241385 but not by DPCPX. We then tested the suppressor potency of isolated Treg39+ together with the enhancer effect of MTX on this potency, and observed that there were no longer differences among ERA, LRA and HC; this further suggests that the differences observed in assays using total Tregs can be attributable to the increased Treg39+ proportions present in ERA. The frequency and function of ERA-R Treg cells were not different from HC or LRA Tregs.Conclusion:The suppressor action of CD39+Tregs is mediated at least in part by adenosine trough A2AR ligation, and the superior suppressive potency of total ERA Tregs is associated with their higher proportion of TregCD39+ cells as compared with HC or LRA. In addition, the augmented suppressor effect observed in the presence of MTX is partly mediated by an increased adenosine production acting on A2AR and is more marked in ERA patients reflecting again their higher proportion of Treg39+ cells. This indicates that MTX cooperates with Treg39+ cells in the control of inflammation.