Background/Purpose: Inhibition of AICAR transformylase by MTX results in augmented release of adenine nucleotides to the extracellular space; these are rapidly hydrolysed by ectonucleotidases CD39 and CD79 rendering the antiinflammatory agent adenosine. 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. Our objective was to study the expression of CD39 on circulating Treg cells of untreated early RA (ERA) patients and its relation with the ex vivo effect of MTX.

Methods: Peripheral blood was drawn from 30 DMARD- and steroid- naïve ERA (disease duration < 24 weeks), 15 longstanding RA patients (LRA, duration > 2 years) and 45 healthy controls (HC). LRA patients were naïve for biologicals and received low-dose weekly MTX. 10 ERA patients who achieved remission 12 months after initiating MTX were re-evaluated (ERA-R). The frequency of Treg cell subsets was determined 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 sorting. The suppressor potency of Tregs was assessed in cocultures with Tresp, at different Treg/Tresp ratios. Proliferation was determined by CFSE dilution and cytokine secretion by ELISA of culture supernatants.

Results: 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 increased in ERA but not LRA. Total ERA Tregs were more potent suppressors of proliferation, TNFα and IFNγ secretion when compared with HC or LRA, and this difference was downmodulated in the presence of adenosine deaminase (ADA), or the adenosine A2A receptor (A2AR) antagonists DMPX (3,7-dimethyl-1-propargylxanthine) or ZM 241385, but not of the adenosine A1 receptor antagonist DPCPX (8-cyclopentyl-dipropylxanthine). When adding MTX, 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. This effect of MTX was significantly abrogated by ADA, DMPX or ZM 241385 but not 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. 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.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/increased-circulating-cd39foxp3cd4-treg-cells-in-early-rheumatoid-arthritis-facilitate-the-antiinflammatory-action-of-methotrexate/