Background/Purpose: FoxP3+ regulatory CD4+ T cells (Tregs) are key to the homeostasis of the immune system. Stressed cells at inflammatory foci release adenine nucleotides to the extracellular space that can act as enhancers of inflammation but are rapidly hydrolysed by the sequential action of ectonucleotidases CD39 and CD73 rendering the antiinflammatory agent adenosine. CD39, the rate-limiting enzyme in this cascade, is highly expressed by a subset of human FoxP3+Tregs (Treg39+). Methotrexate (MTX), the first line of treatment in Rheumatoid Arthritis (RA), inhibits AICAR transformylase which results in an enhanced release of extracellular adenine nucleotides to be metabolized by Treg39+ cells; hence MTX may cooperate with Treg39+ cells in the control of inflammation.

Methods: Peripheral blood was obtained from 72 DMARD- and steroid- naïve ERA patients with a disease duration < 24 weeks and 72 age and gender-matched healthy controls (HC). 33 ERA patients donated blood again 12 months after initiating MTX (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 sorting. The suppressor potency of Tregs was determined in cocultures of isolated Tregs with Tresp. Proliferation was determined by CFSE dilution; cytokine secretion was measured by ELISA of culture supernatants. Disease activity was assessed using the DAS28-ESR score. Low disease activity (LDA) was defined as a DAS28 ≤3.2.

Results: ERA patients demonstrated a superior frequency of circulating Tregs containing increased proportions of Treg39+ cells. Total ERA Tregs were more potent than HC Tregs and MTX further heightened their potency, with greater amplification in ERA vs HC; differences were reduced by adenosine pathway blockade. The potency of isolated Treg39+ and its enhancement by MTX were comparable for ERA and HC suggesting that the differences seen with total Tregs are due to the increased ERA Treg39+ frequency. Basal Treg39+ proportions > 39.3 (p75 of HC) associated with a good 12 month EULAR response [RR 13.4 (2.9-75.6)]. At 12 months, the ERA Treg39+ frequency had decreased to HC levels but its association with the clinical response remained: the cTreg39+ cell frequency observed at 12 months was still significantly higher in those patients who had achieved LDA. This suggests that Treg cell expression of CD39 is upregulated during the initial stages of ERA as a negative feedback mechanism of inflammation.

Conclusion: MTX cooperates with Treg39+ cells and the basal Treg39+ frequency is a predictor of response. Increased circulating Treg39+ cells in untreated ERA would further facilitate the action of MTX thereby providing a slot for prompt MTX initiation.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/increased-cd39foxp3cd4-regulatory-t-cells-in-early-rheumatoid-arthritis-provide-a-slot-for-prompt-initiation-of-methotrexate-and-serve-as-early-biomarkers-of-clinical-response/