Low expression of CD39 on regulatory T cells as a biomarker for resistance to methotrexate therapy in rheumatoid arthritis

Abstract

Rheumatoid arthritis (RA) is an inflammatory autoimmune disease characterized by joint destruction and severe morbidity. Methotrexate (MTX) is the standard first-line therapy of RA. However, about 40% of RA patients are unresponsive to MTX treatment. Regulatory T cells (Tregs, CD4(+)CD25(+)FoxP3(+)) are thought to play an important role in attenuating RA. To investigate the role of Tregs in MTX resistance, we recruited 122 RA patients (53 responsive, R-MTX; 69 unresponsive, UR-MTX) and 33 healthy controls. Three months after MTX treatment, R-MTX but not UR-MTX showed higher frequency of peripheral blood CD39(+)CD4(+)CD25(+)FoxP3(+) Tregs than the healthy controls. Tregs produce adenosine (ADO) through ATP degradation by sequential actions of two cell surface ectonucleotidases: CD39 and CD73. Tregs from UR-MTX expressed a lower density of CD39, produced less ADO, and had reduced suppressive activity than Tregs from R-MTX. In a prospective study, before MTX treatment, UR-MTX expressed a lower density of CD39 on Tregs than those of R-MTX or control (P < 0.01). In a murine model of arthritis, CD39 blockade reversed the antiarthritic effects of MTX treatment. Our results demonstrate that MTX unresponsiveness in RA is associated with low expression of CD39 on Tregs and the decreased suppressive activity of these cells through reduced ADO production. Our findings thus provide hitherto unrecognized mechanism of immune regulation in RA and on mode of action of MTX. Furthermore, our data suggest that low expression of CD39 on Tregs could be a noninvasive biomarker for identifying MTX-resistant RA patients.

Keywords: adenosine; biomarker; ectonucleotidases; methotrexate; rheumatoid arthritis.

Fig. 1.

Increased frequency of circulating Tregs in R-MTX patients. (A and B) Frequency and absolute number of CD4⁺CD25⁺FoxP3⁺ cells from healthy controls (Healthy) (○, n = 22), R-MTX (●, n = 27), and UR-MTX (♦, n = 30) RA patients analyzed by flow cytometry. (C) Representative dot plot showing frequency of CD4⁺CD39⁺ cells from R-MTX and UR-MTX patients. (D) Percentage of CD4⁺CD39⁺ cells from healthy donors (○, n = 16), R-MTX (●, n = 13), and UR-MTX (♦, n = 20). (E) Representative dot plot showing percentage of CD4⁺CD73⁺ cells from R-MTX and UR-MTX patients. (F) Percentage of CD4⁺CD73⁺ cells from healthy donors (○, n = 10), R-MTX (●, n = 13), and UR-MTX (♦, n = 20). (G and H) Frequency of CD4⁺CD25⁺ (G) and CD4⁺CD25⁺FoxP3⁺ (H) cells expressing CD39 in healthy donors (○, n = 22), R-MTX (●, n = 27), or UR-MTX (♦, n = 30). (I) Frequency of CD4⁺CD25⁻ cells expressing CD39 in healthy donors (○, n = 16), R-MTX (●, n = 13), or UR-MTX (♦, n = 20). Horizontal bars = mean; vertical bars = SEM. *P < 0.05.

Fig. 2.

Low CD39 expression, impaired ADO production, and reduced suppressive function of Tregs from UR-MTX patients. (A) Representative histogram of CD39 expression on CD4⁺CD25⁺ cells from healthy individuals, R-MTX, and UR-MTX patients. (B) Mean fluorescence intensity (MFI) of CD39 on CD4⁺CD25⁺ and CD4⁺CD25⁻ cells from healthy donors (n = 10), R-MTX (n = 13), and UR-MTX (n = 20) 3 mo after MTX treatment. **P < 0.01. (C) Representative chromatograms of ADO levels in culture supernatant of Tregs from healthy donors, R-MTX, and UR-MTX patients incubated with ADP. Tregs from healthy donors cultured without ADP serve as control (Blank). (D) Concentration of ADO in the supernatant of CD4⁺CD25⁺ and CD4⁺CD25⁻ cells from healthy individuals (n = 5), R-MTX (n = 6), and UR-MTX patients (n = 6). *P < 0.05. (E) Concentration of Pi in the supernatant of CD4⁺CD25⁺ and CD4⁺CD25⁻ cells of healthy controls (n = 5), R-MTX (n = 6), and UR-MTX (n = 6) patients. *P < 0.05. (F) Representatives histograms of the percentages of proliferation of Teff cells stained with Dye Efluor 670 and incubated with Treg cells from healthy or MTX-treated RA donors at the indicated Treg:Teff ratio. (G) Percentage of suppression of Teff by Tregs from healthy (n = 5), R-MTX (n = 5), and UR-MTX (n = 4) donors, using the formula [Teff only – (Treg + Teff)/Teff only] × 100. *P < 0.05 and **P < 0.01 compared with healthy controls. All data are mean ± SEM.

Fig. 3.

Low CD39 expression density on Tregs in UR-MTX patients before and after MTX treatment. (A) Representative histogram of CD39 expression on the Tregs from R-MTX and UR-MTX patients before and after MTX treatment. (B) MFI of CD39 on CD4⁺CD25⁺FoxP3⁺ Tregs from R-MTX (n = 15) and UR-MTX (n = 11) before (●) and after (▲) MTX treatment. (C) MFI of CD39 on the Tregs from R-MTX (n = 15) and UR-MTX (n = 11) before and after MTX treatment and healthy controls (n = 16). **P < 0.01. (D) MFI of CD39 on CD4⁺CD25⁺ cells from healthy donors (n = 26), R-MTX (n = 28), and UR-MTX (n = 31) after MTX treatment. **P < 0.01.

Fig. 4.

Effect of MTX on AIA. C57BL/6 FoxP3-GFP mice immunized and boosted with mBSA and treated with MTX or vehicle were challenged intraarticular (i.a.) with mBSA or saline. Neutrophils in the joints (A) and intraarticular mechanical hyperalgesia (B) were determined. mBSA-specific IgG concentration in the serum was determined by ELISA (C). Frequency of CD4⁺FoxP3⁺ cells in the spleen was determined by FACS (D). Immunized mice pretreated with MTX or vehicle were injected with anti-CD25 or normal IgG (−) and challenged i.a. with mBSA or saline (Sal). Neutrophils in the joints (E) and mechanical hyperalgesia (F) were determined. Immunized mice pretreated with MTX or vehicle and injected with a CD39 inhibitor (CD39i, ARL67156) or not (−) were challenged i.a. with mBSA or saline. Neutrophils in the joints (G) and mechanical hyperalgesia (H) were determined. Frequency of CD4⁺FoxP3⁺ cells in the spleen was determined by FACS (I). Data represent mean ± SEM (n = 5), representative of two experiments. *P < 0.05.