surface expression of cd39 identifies an enriched treg-cell subset in the rheumatic joint, which...

Eur. J. Immunol. 2014. 44: 2979–2989 ImmunomodulationDOI: 10.1002/eji.201344140 2979

Surface expression of CD39 identifies an enrichedTreg-cell subset in the rheumatic joint, whichdoes not suppress IL-17A secretionJessica Herrath, Karine Chemin, Inka Albrecht, Anca I. Catrinaand Vivianne Malmstrom

Rheumatology Unit, Department of Medicine, Karolinska University Hospital Solna, KarolinskaInstitutet, Stockholm, Sweden

Treg cells are important for the maintenance of self-tolerance and are implicated inautoimmunity. Despite enrichment of Treg cells in joints of rheumatoid arthritis (RA)patients, local inflammation persists. As expression of the ATP-hydrolyzing enzymesCD39 and CD73 and the resulting anti-inflammatory adenosine production have beenimplicated as an important mechanism of suppression, we characterized FOXP3+ Tregcells in blood and synovial fluid samples of RA patients in the context of CD39 and CD73expression. Synovial FOXP3+ Treg cells displayed high expression levels of rate-limitingCD39, whereas CD73 was diminished. FOXP3+CD39+ Treg cells were also abundant insynovial tissue. Furthermore, FOXP3+CD39+ Treg cells did not secrete the proinflamma-tory cytokines IFN-γ and TNF after in vitro stimulation in contrast to FOXP3+CD39− T cells.FOXP3+CD39+ Treg cells could be isolated by CD39 and CD25 coexpression, displayed ademethylated Treg-specific demethylated region and coculture assays confirmed thatCD25+CD39+ T cells have suppressive capacity, while their CD39− counterparts do not.Overall, our data show that FOXP3+CD39+ Treg cells are enriched at the site of inflam-mation, do not produce proinflammatory cytokines, and are good suppressors of manyeffector T-cell functions including production of IFN-γ, TNF, and IL-17F but do not limitIL-17A secretion.

Keywords: Rheumatoid arthritis � Regulatory T cells � FOXP3 � Th17 � CD39

� Additional supporting information may be found in the online version of this article at thepublisher’s web-site

Introduction

Natural Treg cells are crucial for the maintenance of self-toleranceand they suppress the activation, proliferation, and effectorfunctions of several immune cells [1]. Moreover, Treg cells arecharacterized by high surface expression of CD25 and by theintranuclear molecule FOXP3, the lineage marker for this T-cellsubset with central importance for their suppressive program [2].Different mechanisms are implicated by which Treg cells suppressproliferation and cytokine output of target cells, including IL-2

Correspondence: Dr. Vivianne Malmstrome-mail: [email protected]

suppression of T cells, killing of effector cells by granzyme- orperforin-dependent pathways, modifying APC function, and viacytokine secretion, for example, IL-10 or TGF-β [3]. Furthermore,Treg cells play a crucial role in the prevention of autoimmune dis-eases and their impairment or frequency reduction is believed tocontribute to autoimmunity [4].

Rheumatoid arthritis (RA) is a chronic and inflammatory dis-order, which primarily affects the synovial joints and if untreatedleads to bone and cartilage destruction [5]. CD4+CD25bright Tregcells are enriched at the site of inflammation compared to theirpresence in the circulation of RA patients [6–8], and are functionalwith the capacity to suppress autologous effector T cells from bothjoint and peripheral blood (PB) origin [6–8].

C© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.eji-journal.eu

2980 Jessica Herrath et al. Eur. J. Immunol. 2014. 44: 2979–2989

CD39 was originally described as a marker of activated B cells[9] and is expressed on monocytes, DCs, natural killer cells, and asubset of activated T cells [10]. Recently, CD39 expression hasbeen described on FOXP3+ Treg cells and ATP hydrolysis hasbeen suggested to be a novel mechanism of Treg-cell suppression[11]. CD39 is an ectonucleotidase, which in concert with CD73,hydrolyzes ATP or ADP to adenosine [12, 13]. Adenosine executesits anti-inflammatory functions via binding to type 1 purinergiccell surface receptors, A1, A2a, A2B, or A3, and the A2a receptorseems to be the major adenosine receptor expressed on T cells[11]. Binding of adenosine leads to a rise of intracellular cAMPlevels that subsequently suppresses T-cell effector function suchas proliferation and CD25 upregulation [14].

Lately, the role of CD39+FOXP3+ Treg cells has been investi-gated in the context of autoimmunity. In the setting of multiplesclerosis, Fletcher et al. demonstrated that frequencies of circu-lating CD39+FOXP3+ Treg cells were diminished and impairedwhile CD39+FOXP3+ Treg cells of healthy individuals suppressedTh17 responses whereby their CD39− counterparts did not [15]. Injuvenile idiopathic arthritis (JIA), CD4+CD39+ T cells were highlyenriched in synovial fluid (SF) and CD39 expression could distin-guish between a memory T-cell and Treg-cell population [16].

So far, the role of FOXP3+CD39+ Treg cells has not been inves-tigated in RA and it is unclear how frequent these cells are in theperiphery, SF, and inflamed joint tissue, and how efficiently theyfunction in suppressing effector T-cell responses, especially Th17responses.

In this study, we have utilized paired blood and SF samplesof RA patients to investigate the frequencies of CD39 and CD73on CD4+ T cells and on FOXP3+ Treg cells. Further, we exam-ined the suppressive function of synovial CD4+CD25+CD39+ andCD4+CD25+CD39− T cells. We demonstrate that synovial inflam-mation associates with local enrichment of CD39+ Treg cellsthat could efficiently suppress effector T-cell proliferation but notIL-17A secretion.

Results

Synovial CD4+ T cells express elevated CD39 anddiminished CD73 levels

As CD39 expression was shown to be upregulated at the siteof inflammation in patients with JIA [16], we investigated theexpression of CD39 and CD73 on the CD4+ T-cell population inboth PB and SF of patients with RA and in SF of patients with anky-losing spondylitis (SpA) (Fig. 1A). As can be seen in Figure 1B,CD4+CD39+ T cells are significantly enriched (p < 0.0001) in thejoints of RA patients compared to PB of healthy controls (HCs) andRA patients (35.4 ± 17.3% in RA-SF compared to 7.4 ± 2.3% inHC-PB or 6.2 ± 3.3% in RA-PB), and the same trend was seenfor SpA (23.5 ± 13.5%). In addition to the higher frequencyof CD39 on synovial CD4+ T cells, the MFI was also higher forSF of RA and SpA patients compared to blood of HCs and RApatients (p < 0.0001) (Fig. 1C). In contrast, CD73 expression on

CD4+ T cells was significantly reduced (p < 0.0001) in SF of RApatients compared to blood of HCs and RA patients (6.2 ± 3.7% inRA-SF compared to 15.8 ± 6.5% in HC-PB or 19.9 ± 8.9% inRA-PB), the same pattern is again seen for SpA-SF (10.2 ± 4.9%)(Fig. 1D). Despite the fact that the proportion of CD73 withinsynovial CD4+ T cells was lower, the MFI showed no significantdifferences between blood and the inflamed joint (Fig. 1E). Insummary, the results show that CD39 is expressed more frequentlyand at a higher protein level on synovial CD4+ T cells comparedto blood, whereas CD73 levels are reduced.

Synovial FOXP3+ Treg cells downregulate CD73whereas CD39 is upregulated

As Treg cells can express CD39 and CD73 and the resulting adeno-sine production contributes to suppression [11, 17], we assessedthe proportion of these enzymes on CD4+FOXP3+ Treg cells(gating strategy in Supporting Information Fig. 1). Figure 2Ashows that CD39 expression on FOXP3+ Treg cells from SF wassignificantly upregulated (p = 0.0007) compared to that on RA-PB(70.1 ± 24.8% in RA-SF and 71.1 ± 24.5% in SpA-SF comparedto 38.9 ± 15.5% in RA-PB). Additionally, the level of expression,that is MFI, of CD39 on synovial CD4+FOXP3+ Treg cells wasalso higher as compared to that in PB (p < 0.0001) (Fig. 2A).Comparable to what was observed on total CD4+ T cells, CD73expression was significantly diminished (p < 0.0001) on synovialFOXP3+ Treg cells compared to that in PB (4.7 ± 2.6% in RA-SFand 2.5 ± 0.9% in SpA-SF compared to 9.2 ± 2.9% in RA-PB and7.7 ± 4.5% in HC-PB) (Fig. 2B) and here the protein levels per cellof CD73 on FOXP3+ T cells were reduced (p < 0.0001) (Fig. 2B).

FOXP3+ T cells in synovial tissue coexpress CD39

The significant upregulation of CD39 on synovial CD4+FOXP3+

T cells of patients with RA and SpA prompted us to study theexpression pattern of CD39 on FOXP3+ T cells in synovial tissueof RA patients. A number of FOXP3+CD39+ T cells were locatedin a large T-cell infiltrate surrounding a vessel (Fig. 2C and Sup-porting Information Fig. 2). Furthermore, quantification of CD39expression on infiltrating FOXP3+ and FOXP3− T cells confirmeda general positivity of CD39 expression, which was comparablebetween the T-cell subsets (Fig. 2D).

FOXP3+CD39+ Treg cells produce minor amounts ofIL-17A, IFN-γ, or TNF compared to CD39− Treg cells

We have previously demonstrated that FOXP3+ Treg cells canproduce minor amounts of proinflammatory cytokines aftershort TCR cross-linking [18], so we assessed their potentialto secrete IL-17A, IFN-γ, and TNF in the context of CD39expression (Fig. 3A). Following stimulation of isolated CD4+

T cells with anti-CD3/anti-CD28 beads, a significant higher


Eur. J. Immunol. 2014. 44: 2979–2989 Immunomodulation 2981

Figure 1. Phenotypic characterization of CD39 and CD73 expression on CD4+ T cells in peripheral blood (PB) of healthy controls, PB and synovialfluid (SF) of RA patients, and SF of SpA patients. (A) Representative dot plots of combined CD39 and CD73 staining are shown of PB from healthydonors and RA patients, as well as SF from RA and SpA patients. Numbers in each quadrant indicate the frequencies of CD39+ or CD73+ cellsamong gated CD4+ T cells. Each dot plot is representative of >10 independent experiments each performed with different donor/patient samples.(B) The frequency of CD39 expression among CD4+ T cells of PB from healthy controls (n = 11), paired PB and SF of RA patients (n = 15), and SF ofSpA patients (n = 10) are also summarized; mean value is indicated by the horizontal line. (C) In comparison to the frequency data, the intensity,that is MFI, of CD39 in the same CD4+ T cells is shown, for control PB, RA-PB, SF from RA, and SpA (n = 11, 15, 15, 10, respectively). Data are shownas median (middle line of the bar) and the minimum and maximum are indicated by the vertical lines. (D) The frequency of CD73 among gatedCD4+ T cells, as well as (E) the MFI, are shown for control PB, RA-PB, SF from RA, and SpA (n = 11, 15, 15, 10, respectively). Kruskal–Wallis test withDunn’s multiple comparison test was performed for (B–E), ***p < 0.0001, **p < 0.01, *p < 0.05.

production of IFN-γ and TNF was observed in FOXP3+CD39−

Treg cells compared to that by FOXP3+CD39+ Treg cells(p = 0.0009 and p = 0.0028, respectively) (Fig. 3B and C).Additionally, the MFI for IFN-γ and TNF was also brighterin the FOXP3+CD39− T-cell compartment (p = 0.0083 andp = 0.0399, respectively) (Fig. 3E and F). No difference wasseen regarding IL-17A production by FOXP3+ Treg cells basedon CD39 expression (Fig. 3D), however the MFI was brighterfor the cytokine output derived from the FOXP3+CD39− T cells(p = 0.0281) (Fig. 3F). The same pattern of cytokine output wasseen for stimulations with PMA and ionomycin (data not shown).Taken together, the absence of CD39 on FOXP3+ Treg cells seemsto indicate cytokine-producing capacity of FOXP3+ Treg cells.

CD25+CD39+ Treg cells are enriched at the site ofinflammation and contain a high frequency of FOXP3

It is well documented that CD4+CD25bright Treg cells are enrichedat the site of inflammation in RA [6–8] and in JIA [19], hencewe investigated the combination of CD25 with CD39 (Fig. 4A)and how those surface markers relate to FOXP3 frequency. Anal-ysis of CD25 expression with CD39 on CD4+ T cells revealed asignificant increase (p = 0.0001) of double-positive T cells when

derived from the joint as compared to RA-PB (4.2 ± 2.9% inRA-SF and 6.5 ± 4.5% in SpA-SF compared to 1.1 ± 0.8% in RA-PB) whereby SpA-SF showed a higher frequency of CD25+CD39+

T cells (Fig. 4B). Moreover, the frequencies of CD25+CD39− T cellswere increased (p = 0.0028) in RA-PB and in SpA-SF compared toHC-PB (0.9 ± 0.5% in RA-PB and 1.1 ± 0.6% in SpA-SF com-pared to 0.4 ± 0.3% in HC-PB) (Fig. 4B). Next, we assessedthe FOXP3 frequency within CD25+CD39− and CD25+CD39+

T cells, which was consistently higher in CD25+CD39+ T cellsthan in their negative counterparts as shown in Figure 4C (HC-PB,p = 0.0010; RA-PB, p < 0.0001; RA-SF, p < 0.0001; and SpA-SF,p = 0.0039).

CD25+CD39+ T cells show demethylated CpG regionsin the TSDR

To exclude that mainly activated cells account for the highFOXP3 expression seen in CD25+CD39+ T cells, we investigatedthe methylation status of the Treg-specific demethylated region(TSDR, Fig. 5A). Sorted synovial CD25+CD39+ T cells (Fig. 5B) ofRA patients revealed a fully demethylated TSDR in three of fourpatients, whereas as expected CD25−CD39− T cells were fullymethylated in that region as can be seen in Figure 5C.



Figure 2. CD39 is upregulated whereas CD73 is downregulated on CD4+FOXP3+ Treg cells. (A) The frequency of CD39 on gated CD4+FOXP3+ Tregcells for control PB, RA-PB, SF from RA, and SpA (n = 11, 15, 15, 10, respectively) is depicted in the top panel, and the mean values are indicated by ahorizontal line. The MFI for the same groups (HC-PB = 11, RA-PB = 15, RA-SF = 15, SpA-SF = 10, respectively) is shown in the bottom panel. Data areshown as median (middle line of the bar) and the minimum and maximum are indicated by the vertical lines. (B) The frequency of CD73 on gatedCD4+FOXP3+ Treg cells is shown (top), as well as for the MFI of CD73 for the CD4+FOXP3+ compartment (bottom). Graphs summarize data fromperipheral blood of controls and RA patients, and SF from RA and SpA patients (n = 11, 15, 15, 10, respectively). Kruskal–Wallis test with Dunn’smultiple comparison test done for (A) and (B), ***p < 0.0001, **p < 0.01, *p < 0.05. (C) Frozen sections of human synovial tissue from RA patients werestained for CD3 (blue), FOXP3 (red), and CD39 (green) and a representative staining depicts CD3+ human T cells expressing FOXP3 and CD39. Scale,20 μm. The staining is representative for three independent experiments with different donors. Single and merged immunofluorescences of atriple-positive T cell are shown to the right. (D) The corrected total cell fluorescence (CTCF) for CD39 was calculated and the resulting quantificationof CD39 expression on FOXP3+ or FOXP3− T cells is displayed (n = 40 cells, for each RA patient and condition). Bars represent mean + SD. Wilcoxonsigned-rank test, n.s.

CD25+CD39+ T cells are anergic and showsuppressive capacity in vitro

Given the different frequency of FOXP3 in CD25+CD39− andCD25+CD39+ T cells, we investigated their functional capacity toinhibit effector T-cell proliferation. Classical coculture assays witheither CD4+CD25+CD39− (I) or CD4+CD25+CD39+ (II) SF T cells(Fig. 6A) revealed that suppression of CD4+CD25−CD39− T effec-tor cell (Teff, III) proliferation at a Treg:Teff ratio of 1:1 was onlymediated by the CD4+CD25+CD39+ T cells (p = 0.0313) but notby their negative counterparts (p = 0.39) (Fig. 6B). Interestingly,CD4+CD25+CD39− T cells did proliferate in vitro in responseto CD3 and APC stimulation as compared to CD4+CD25+CD39+

T cells (p = 0.026) (Fig. 6B). Next, we wanted to explore to whatdegree CD39 and the resulting breakdown of ATP contributes tosuppressive capacity, therefore we setup parallel coculture assayswith CD4+CD25+CD39+ T cells either with or without addedARL-67156, a selective ATPase inhibitor. A tendency of loss ofsuppressive capacity was detected at a Treg:Teff ratio of 0.125:1(p = 0.0625) (Fig. 6C) indicating that ATP hydrolysis is not thesole mechanism of suppression in coculture assays, but additionalmechanisms are likely to contribute.

CD25+CD39+ T cells do not suppress IL-17A secretion

Since CD25+CD39+ and CD25+CD39− T-cell populations exhib-ited different capabilities of suppressing effector T-cell prolif-eration, we subsequently explored their abilities in suppressingcytokine secretion, with a special emphasis on Th17 responses.Therefore, the supernatants of the coculture experiments werescreened not only for the presence of IL-17A, but also the relatedcytokines IL-17F, IL-22, and for other T-cell subset signaturecytokines, that is, IL-10, IL-13, IFN-γ, and TNF. The cytokinesecretion of the coculture wells containing Teff cells and Tregcells (either CD25+CD39− or CD25+CD39+) were compared tothe cytokine secretion of the wells containing Teff cells alone.The ratio (cytokine secretion of the coculture wells divided by thesecretion of the Teff wells alone) was used as a degree of suppres-sion and expressed as percentage of secretion of CD25− Teff cells.As can be seen in Figure 6E, synovial CD25+CD39+ Treg cellssuppressed the secretion of IL-17F, IL-22, IL-10, IL-13, IFN-γ, andTNF by 50% (as indicated by the dotted line) or more, while IL-17Aproduction was not suppressed at all. Of note, the same tendencywas also observed for blood-derived CD25+CD39+ Treg cells ofRA patients, whereas IL-17A secretion could not be detected in



Figure 3. Cytokine secretion profile of FOXP3+CD39+ and FOXP3+CD39− T cells of paired PB and SF RA samples after in vitro stimulation.(A) Representative intracellular staining for IFN-γ, TNF, and IL-17A in PB and SF of CD4+FOXP3+ T cells. The frequency of FOXP3+CD39+ andFOXP3+CD39− T cells that were positive for IFN-γ, TNF, or IL-17A is indicated. Staining is representative of six independent experiments, eachperformed with different donors. (B–D) A summary of (B) IFN-γ+ cells, (C) TNF+ cells, and (D) IL-17A+ positive cells among FOXP3+CD39− T cellsand FOXP3+CD39+ T cells from PB (n = 6, white bars) and SF (n = 6, black bars) is shown. (E–G) The intensity (MFI) of the (E) IFN-γ, (F) TNF, and(G) IL-17A stainings in FOXP3+CD39− T cells and FOXP3+CD39+ T cells from PB (n = 6, white bars) and SF (n = 6, black bars) is depicted. Barsrepresent mean + SD. Two-way ANOVA test (B–G), ***p < 0.0001, **p < 0.01, *p < 0.05.

assays with blood-derived Teff cells of age- and sex-matched HCs(Supporting Information Fig. 3).

Strikingly, synovial CD25+CD39− T cells failed to suppress thecytokines studied (Fig. 6D). Indeed, when looking at the cytokineproduction by either CD25+CD39− or CD25+CD39+ T cells alone,it became obvious that the CD25+CD39− population secretedcytokines instead of suppressing them, mainly IL-17A(214 ± 341 pg/mL by CD39− Treg cells compared to2.8 ± 2.7 pg/mL by CD39+ Treg cells), IL-17F (423 ± 588 pg/mLby CD39− Treg cells compared to 0.2 ± 0.5 pg/mL by CD39+

Treg cells), IFN-γ (318 ± 407 pg/mL by CD39− Treg cellscompared to 49.5 ± 116 pg/mL by CD39+ Treg cells), andTNF-α (109 ± 104 pg/mL by CD39− Treg cells comparedto 16.3 ± 19.0 pg/mL by CD39+ Treg cells) (Fig. 6F).Furthermore, when comparing the cytokine production ofCD25+CD39− T cells with the production of CD25− Teff cells,we observed that CD25+CD39− T cells produce higher amountsof IL-17A (214 ± 341 pg/mL by CD39− Treg cells compared to23 ± 49 pg/mL by CD25− Teff cells) and IL-17F (423 ± 588 pg/mLby CD39− Treg cells compared to 100 ± 234 pg/mL by CD25−

Teff cells), but less IFN-γ (318 ± 407 pg/mL by CD39− Tregcells compared to 835 ± 678 pg/mL by CD25− Teff cells) andTNF-α (109 ± 104 pg/mL by CD39− Treg cells compared to402 ± 323 pg/mL by CD25− Teff cells).

In conclusion, CD25+CD39+ Treg cells do not suppress IL-17Asecretion in SF and PB of RA patients, whereas suppression ofIL-17F, IL-22, IL-10, IL-13, IFN-γ, and TNF was efficiently done. Instriking contrast, synovial CD25+CD39− T cells failed to suppressany cytokine production but instead produced cytokines.

Discussion

Accumulation of Treg cells at the site of inflammation in auto-immune diseases is well documented [7, 8, 19–22], however,despite this enrichment, inflammation is ongoing and persists ina chronic or at least recurrent way. Nevertheless, synovial Tregcells have been shown to be suppressive (in vitro) and exert theirfunction by limiting proliferation and cytokine production of effec-tor T cells [6, 8]. Suppressive capacity of Treg cells is mediated



Figure 4. Comparison of the frequency of CD25+CD39− and CD25+CD39+ T cells between PB and SF mononuclear cells in healthy controls andpatients with RA or SpA. (A) Representative dot plots of CD25 and CD39 staining in HC-PB, RA-PB, RA-SF, and SpA-SF is displayed. Data were gatedon CD4+ T cells and numbers indicate the frequencies of CD25+CD39−, CD25+CD39+, and CD25−CD39+ T cells among gated CD4+ T cells. (B) Asummary for the expression of CD25 on CD39− T cells (left graph) and CD39+ T cells (right graph) within all the cohorts investigated, n = 11, 15,15, 10, respectively, for HC-PB, RA-PB, RA-SF, and SpA-SF is shown and the mean values are indicated by a horizontal line. Kruskal–Wallis testwith Dunn’s multiple comparison test, ***p < 0.0001, **p < 0.01, *p < 0.05. (C) The frequency of FOXP3 expression in CD25+CD39− and CD25+CD39+

T-cell subsets was analyzed for HC-PB (n = 11, upper left), RA-PB (n = 15, upper right), RA-SF (n = 15, lower left), and SpA-SF (n = 10, lower right).Wilcoxon signed-rank test, ***p < 0.0001, **p < 0.01.

by a number of different mechanisms and lately CD39 expressionon FOXP3+ Treg cells, which results together with CD73 in anti-inflammatory adenosine production, has been suggested to be anadditional one [11].

From this study, we can conclude that CD39 expression wassignificantly increased in the rheumatic joint, both on the wholeCD4+ T-cell population, as well as on FOXP3+ Treg cells ascompared to blood of both patients and healthy donors. Wefurther demonstrate an abundance of FOXP3+CD39+ T cellsin the rheumatic synovia emphasizing that also at the site oftissue destruction this Treg-cell subset is present. Elevated CD39expression has previously been reported for CD4+ T cells in SF ofchildren with JIA [16]. Upregulation of CD39 expression has beendocumented in other disease settings such as cancer [23, 24] andfollowing virus infection [25, 26], indicating that inflammationor the local microenvironment itself influences CD39 expression.In murine settings, CD39 upregulation has been demonstrated onCD4+ T cells upon activation [27] and under hypoxic conditions[28].

CD39 provides the rate-limiting step for breakdown of ATP,followed by CD73 that converts AMP to adenosine. In our study,we saw a decrease of CD73 expression on CD4+ T cells andon FOXP3+ Treg cells at the site of inflammation, suggesting areduced production of adenosine. A similar expression patternhas also been reported for JIA [16] and we hypothesize that thelocal environment at the site of inflammation could account forthis, since CD73 is downregulated upon activation. Additionally,inflammatory cytokines such as IL-4, IL-12, IL-21, and IFN-γ cancounteract upregulation of CD73 [27]. Furthermore, the activityof adenosine deaminase, the enzyme that catalyzes the deamina-tion of adenosine to inosine, was specifically increased in rheuma-toid synovial fibroblasts and SF, which limits even more the anti-inflammatory effect of adenosine [29].

Several reports demonstrated that CD39 expression on CD4+

T cells can distinguish between a regulatory and an effector mem-ory T-cell population in mouse [30] and in human [16, 31]. Toclarify the function of CD39 on CD4+FOXP3+ Treg cells in aninflammatory setting, we stimulated CD4+ T cells from blood



Figure 5. TSDR demethylation of sorted synovial CD25+CD39+ Treg cells. (A) Overview of the TSDR position within the FOXP3 gene locus (adaptedfrom [39]). (B) Representative dot plot depicting the sorting strategy for subsequent methylation analysis of CD25−CD39− and CD25+CD39+ T cellsas indicated by each box. FOXP3 frequency of CD25−CD39− and CD25+CD39+ T cells (left and right histogram, respectively) is shown. Numbersindicate the mean of all experiments, n = 4. (C) Methylation pattern of the TSDR in CD25−CD39− and CD25+CD39+ T cells. Fifteen individualCpG islands within the TSDR were analyzed and are represented by the horizontal lines. The degree of methylation is color-coded according tothe scale on the left, from blue (100% methylation) to yellow (0% methylation). Percentages show the mean methylation within the TSDR. Onerepresentative experiment is shown for CD25−CD39− T cells, and all independent experiments are shown for CD25+CD39+ T cells (n = 4).

and SF of RA patients and investigated the cytokine output ofFOXP3+CD39+ and FOXP3+CD39− T cells. The absence of CD39on CD4+FOXP3+ Treg cells was an indicator for cytokine produc-tion, since FOXP3+CD39− T cells showed a higher percentage andprotein per cell level (MFI) of both IFN-γ and TNF compared totheir positive counterparts. Hence, FOXP3+CD39+ T cells repre-sented a classical Treg-cell phenotype.

Given the differential pattern in cytokine secretion we wereprompted to study the functional capacities of the subsets.Therefore, we characterized the dual expression of CD39 andCD25 in healthy individuals and in patients with RA or SpA.We observed an accumulation of CD25+CD39+ T cells in theinflamed joint either from RA or SpA patients compared to bloodderived from RA patients or HCs. Furthermore, the double-positiveT cells expressed significantly more FOXP3, again pointing outto CD39 expression being important for a regulatory phenotype.Subsequently, methylation analysis could verify that the FOXP3population found in synovial CD25+CD39+ T cells consisted of“true Treg cells,” as the TSDR was fully demethylated (0% methy-lation) for the majority of patients tested, with one outlier demon-strating an intermediate phenotype.

To validate these results, we performed conventional cocultureassays to assess the functionality of the subsets. Our results showthat in contrast to synovial CD25+CD39+ T cells, their negativecounterparts were not able to suppress proliferation but insteadwere proliferative. As Treg cells are normally refractory to TCRstimulation and show anergic properties in vitro, this observa-tion might be an indication for the importance of CD39 expres-sion for regulatory function. In line with this, Dwyer et al. couldshow that CD25+CD39− T cells isolated from human PB prolif-erated and secreted cytokines in large amounts. Furthermore,under Th17 promoting conditions, CD25+CD39− T cells expressedIL-17A and IFN-γ [31]. In contrast, Fletcher et al. demonstratedthat both CD25+CD39− T cells and CD25+CD39+ T cells fromhealthy donors were equally capable in suppressing prolifera-tion and IFN-γ secretion, while IL-17 could only be efficientlysuppressed by CD25+CD39+ T cells [15].

In our study, it became obvious that synovial CD25+CD39−

T cells were not able to suppress any cytokine output butinstead produced IL-17A/F, IFN-γ, and TNF in large amounts. Incontrast, synovial CD4+CD25+CD39+ T cells were able to sup-press most Th1, Th2, and Th17 cytokines by 50% or more but



Figure 6. Synovial CD25+CD39+ Treg cells are able to suppress effector T-cell proliferation but do not limit IL-17A secretion. (A) Representativeflow cytometry dot plot showing the sorting strategy for subsequent coculture experiments of synovial fluid samples, population I (CD25+CD39−

T cells), population II (CD25+CD39+ T cells) sorted as Treg cells and population III (CD25−CD39− T cells) representing effector T cells. Sortingstrategy is representative for six independent experiments with different donors. (B) Synovial CD4+CD25−CD39− T cells isolated from six patientswere cultured alone or in the presence of either CD25+CD39− or CD25+CD39+ Treg cells. Proliferation was assessed by cpm of CD25+CD39− Tregcells (white bar), CD25+CD39+ Treg cells (dark gray bar), CD25−CD39− Teff cells (black bar) alone, or the combination of CD25− Teff cells witheither CD39− Treg cells (dark gray bar) or CD39+ Treg cells (light gray bar) (n = 6). All values are expressed as mean + SD. Wilcoxon signed-rank test, *p < 0.05. (C) Synovial CD25+CD39+ Treg cells (n = 6) were cultured alone or in the presence of 100 μg/mL ARL-67156, a selective ATPanalogue and percentage of suppression by CD25+CD39+ Treg cells is depicted. Wilcoxon signed-rank test, n.s. (D, E) Cell culture supernatantswere collected from the different cocultures performed with (D) CD25+CD39− Treg cells or (E) CD25+CD39+ Treg cells and were analyzed for sevendifferent cytokines: IL-17A, IL-17F, IL-22, IL-10, IFN-γ, TNF, and IL-13 (n = 6, same group as for proliferation data in B and C). Data are presentedas percentage of secretion of CD25− Teff cells, that is, the secretion of cytokines in the cocultures as compared to secretion by CD4+CD25− Teffcells alone and the ratio was calculated. Dotted line at 100 indicates that Treg cells + Teff cells (coculture wells) secreted 100% of the amount ofcytokines produced by the CD25− Teff cells alone, that is, no suppression of cytokine secretion by Treg cells. Dotted line at 50 indicates that Tregcells + Teff cells (coculture wells) secreted 50% of the amount of cytokines produced by the CD25− Teff cells alone, that is, 50% suppression ofcytokine secretion by Treg cells in the coculture. Bars represent mean + SD. (F) Cytokine secretion of CD25+CD39− (black bars), CD25+CD39+ Tregcells (white bars), and CD25− Teff cells (gray bars) alone was analyzed (n = 6). Bars represent mean + SD.

strikingly did not suppress IL-17A secretion efficiently. Notably,a similar tendency could be observed for CD4+CD25+CD39+

T cells derived from PB of RA patients. The obvious discrepancy ofCD4+CD25+CD39+ T cells being capable of suppressing the secre-tion of IL-17F but not IL-17A could be explained by, for example,the differential chromatin regulation mechanisms of IL-17A andIL-17F [32, 33]. IL-17 suppression by Treg cells has been a matterof discussion since it was shown that Treg cells suppress more eas-ily Th1 or Th2 responses, but not Th17 responses [34] and also inan earlier study of ours, we could not find a consistent pattern ofIL-17A suppression [18]. Now, revisiting this issue with a specificfocus on the CD39+FOXP3+ Treg-cell subset, it became appar-ent that CD39+ Treg cells might indirectly contribute to diseasepathogenesis, as IL-17 is a cytokine that enhances joint inflamma-tion and destruction in RA [35].

The immunosuppressive mechanism executed by CD39+ Tregcells is first the removal of ATP by hydrolysis, which alreadyreduces its proinflammatory actions including the production ofIL-6 and TNF [36], and second the production of adenosinetogether with CD73 [11, 17], which subsequently suppresses dif-

ferent T-cell effector functions [14]. Hence, we inhibited CD39activity by adding the ATP analogue ARL-67156 to the performedcocultures to see if the inhibition is reversible. We saw less sup-pression with decreasing numbers of CD4+CD25+CD39+ T cellsadded to the culture, however suppressive function could not betotally abrogated, which might be an indication for additional sup-pressive mechanisms taking place in vitro, for example, cytokinesecretion, IL-2 depletion, modification of APCs, etc. [3]. Consis-tent with our observation, in the setting of healthy donors neitherinhibition of CD39 activity nor an A2A receptor antagonist couldreverse suppressive function mediated by CD39+ Treg cells[15].

In conclusion, our data demonstrate that CD39 expression onCD4+FOXP3+ T cells can distinguish between two distinct subsetswith different functionalities and that the FOXP3+ T-cell subsetexpressing CD39 resembles the classic Treg-cell phenotype.

Moreover, we provide evidence that in RA CD25+CD39+ Tregcells do not suppress IL-17A secretion. Hence, this populationcould indirectly contribute to disease pathogenesis and this partlyexplains why accumulation of Treg cells is seen at the site of



inflammation without facilitating it. Further studies are needed tobetter understand the function of CD39+ Treg cells in healthy con-ditions and more specifically the mechanism behind the differen-tial suppression of cytokine secretion exhibited by CD25+CD39+

Treg cells in RA with a special focus on Th17 responses. Takentogether, our data support the idea that specific enhancing ofIL-17A suppression abilities of CD39+ Treg cells and/or moregeneral IL-17 blocking strategies, such as anti-IL-17 antibodies[37] represent potential therapeutic strategies to control synovialchronic inflammation.

Materials and methods

Samples from patients with RA

PB and SF samples were taken at the Rheumatology Clinic atKarolinska University Hospital, Sweden at times of active inflam-mation (relapses) that required joint effusions. Mononuclearcells were isolated by Ficoll separation (Ficoll-Plaque Plus, GEHealthcare, Uppsala, Sweden) and cryopreserved in liquid nitro-gen until use. A total of 34 patients with RA (24 female, 10 male;17 CCP+, 17 CCP−) with a mean age of 59.6 (range: 26–87)at time of sampling were included in the study together withten patients with SpA (ten males with a mean age of 49.7) and15 HCs. Since SF cannot be obtained from healthy donors, weutilized SF from another rheumatic disease, SpA as a “diseasecontrol” for the RA SF samples. Fifteen RA, ten SpA patients, andthe HC samples were used for phenotypic analysis, and four of theRA patient samples were also included in the intracellular cytokineanalysis (n = 6). For coculture assays, ten RA patient samples andfour HCs; for confocal microscopy, three patient samples; and forthe TSDR methylation analysis, four patient samples were used.A detailed description of the patient characteristics can be foundin Supporting Information Table 1. All RA patients attended theRheumatology Clinic at Karolinska University Hospital on a reg-ular basis and fulfilled the American College of Rheumatologycriteria for RA. The study was performed with ethical approval ofthe Local research ethics committee at Karolinska University Hos-pital and all research subjects gave informed consent according tothe declaration of Helsinki.

Flow cytometry

Single-cell suspensions were stained with anti-human mAb spe-cific for CD3 (clone UCHT-1), CD4 (SK3, RPTA-4), CD14(MphiP9), CD25 (2A3), CD39 (A1), CD73 (AD2), FOXP3(206D), IL-17A (BL168), IFN-γ (4S.B3), TNF-a (Mab11) conju-gated to FITC, PE, PerCP, PerCP-Cy5.5, PE-Cy7, Alexa-Fluor647,Alexa-700, allophycocyanin-Cy7, allophycocyanin-H7, BrilliantVi-olet421, and purchased from either BD Biosciences (San Jose,CA, USA) or Biolegend (San Diego, CA, USA). LIVE/DEAD NearInfra-Red Dead Cell Stain (Invitrogen) was used to ascertain cell

viability. A total of 1 × 106 PBMCs or SFMCs were surface-stainedin 96-well plates for 20 min at 4°C and stained intracellular orintranuclear. Data were acquired on a Gallios (Beckman Coulter,Brea, CA, USA) and analyzed with FlowJo software, version 9.3.1for Mac (Treestar, Ashland, OR, USA).

Intranuclear/intracellular staining

Intranuclear staining for FOXP3 was done according to man-ufacturer’s instructions using the FOXP3/Transcription factorstaining buffer set (eBioscience, San Diego, CA, USA). Forintracellular cytokine staining, 1 × 105 MACS negativelysorted CD4+ T cells (Milteny-Biotec, Bergisch-Gladbach, Ger-many) were incubated at 37°C, either unstimulated, in thepresence of 10 ng/mL PMA, 1 μg/mL ionomycin (Sigma-Aldrich),or in the presence of CD3/CD28 Dynabeads (bead-to-cell ratioof 1:1, Dynal, Invitrogen) for 5 h and with 10 μg/mL BrefeldinA (Sigma-Aldrich) in the last 4 h of incubation. Cells werefirst surface-stained, fixed, and permeabilized as for intranu-clear FOXP3 staining (eBioscience), and subsequently stained forFOXP3, IL-17A, IFN-γ, and TNF for 30 min at 4°C.

Cell sorting

Cells were fluorescently labeled for CD3, CD4, CD25, and CD39and sorted in three T-cell populations: CD25−CD39− effectorT cells, CD25+CD39+, and CD25+CD39− T cells (in SF only) usingan Influx (BD Biosciences). CD3neg cells were sorted and usedafter irradiation (30 Gy) as APCs. Sorted cells were analyzed forpurity (sort purity was greater than 95% for all sorted subpop-ulations) and CD25+CD39− and CD25+CD39+ Treg cells wereanalyzed for FOXP3 expression (FOXP3 expression, average forall experiments of SF, 52%, 75%, respectively). FOXP3 expressionof blood-derived CD25+CD39+ Treg cells for HCs was 98.7% andfor RA patients 96.7%.

Coculture and cytokine assay

Coculture experiments (n = 10) were setup with 3 × 104 irradi-ated autologous CD3neg APCs and 1 × 104 CD25−CD39− effec-tor T cells alone or with different ratios of CD25+CD39+/− Tregcells (ratio Teff:Treg; 1:1 to 1:0.125). Cells were stimulated withplate-bound anti-CD3 (0.5 μg/mL, clone OKT3) cultured in RPMI-1640 supplemented with 5% heat-inactivated serum, penicillin(100 U/mL), streptomycin (100 μg/mL), 2 mM L-glutamine, and10 mM Hepes. CD25+CD39+ Treg cells were cultured in thepresence or absence of the ATPase inhibitor ARL-67156 (100 μM,Sigma-Aldrich). Cells were incubated at 37°C for 6 days, the last15–18 h in the presence 1 μCi of (3H) thymidine. Cytokines incell culture supernatants were detected using the Bio-Plex TMcytokine detection array (Bio-Rad, Hercules, CA, USA). The panel



included IL-10, IL-13, IL-17A, IL-17F, IL-22, IFN-γ, and TNF andwas run according to manufacturer’s instructions.

Confocal microscopy

Frozen sections of synovial tissue were permeabilized with0.1% saponin and stained with CD39 Alexa-Flour488 (clone A1,Invitrogen), FOXP3 Alexa-Flour647 (clone 206D, Biolegend), andCD3 BrilliantViolet421 (clone UCHT1, Biolegend) for 1 h at 37°C.Sections were mounted with Fluoromount-G (SouthernBiotech).Images were acquired using a Leica TCS SP5 and acquisitionwas performed using a 63× oil objective. A z-dimension serieswas taken every 0.2 μm. Images were analyzed with the ImageJsoftware. Quantification of CD39 staining of FOXP3+ and FOXP3−

T cells was done as described elsewhere [38].

TSDR methylation analysis

SF cells from male RA patients (n = 4) were sorted intoCD25−CD39− and CD25+CD39+ T cells. Sorted cells were ana-lyzed for purity (sort purity was greater than 95% for all sortedsubpopulations) and CD25+CD39+ Treg cells were analyzed forFOXP3 expression (FOXP3 expression, RA#1: 77.2%; RA#2:92.8%; RA#3: 61.5%; RA#4: 66.9%; average for all experiments74.6%). Genomic DNA was isolated according to manufacturer’sinstruction using the DNeasy Blood and tissue kit (Qiagen, Hilden,Germany). DNA methylation studies of 15 CpG motifs withinthe TSDR (Amplicon 5) were done by bisulphite sequencing asdescribed elsewhere ([39]) and were performed by Epiontis.

Statistical analyses

All statistical analyses were performed by using Prism 5 software(Graph Pad, San Diego, CA, USA). Comparisons between three ormore groups were made using one-way ANOVA, Kruskal–Wallistest with Dunns multiple comparison test, comparisons betweenpaired samples were made using Wilcoxon signed-rank test;p values less than 0.05 were considered statistically significant.If not otherwise indicated, data are presented as mean with SD.

Acknowledgements: The authors thank staff and patientsat the Rheumatology Clinic of Karolinska University Hospi-tal, Gull-Britt Almgren, Julia Bostrom, Gloria Rostvall, and EvaJemseby for organizing the sampling, storage, and administra-tion of biomaterial, Marianne Engstrom for excellent technicalassistance, and Annika van Vollenhoven for excellent cell sorting.This study is supported by grants from the Swedish Associationagainst Rheumatism, the King Gustaf V 80-year Foundation, the

TREG CENTER consortia, EU-FP7 project Masterswitch (HEALTH-F2-2008-223404), IMI-JU funded project BTCure 115142-2,Swedish Foundation for Strategic Research, FP7-HEALTH-2012-INNOVATION-1 Euro-TEAM (305549-2), Initial TrainingNetworks 7th framework program Osteoimmune (289150), andthe Swedish Research Council.

Conflict of interest: The authors declare no financial or commer-cial conflict of interest.

References

1 Sakaguchi, S., Naturally arising CD4+ regulatory T cells for immuno-

logic self-tolerance and negative control of immune responses. Annu.

Rev. Immunol. 2004. 22: 531–562.

2 Hori, S., Nomura, T. and Sakaguchi, S., Control of regulatory T cell devel-

opment by the transcription factor Foxp3. Science 2003. 299: 1057–1061.

3 Sakaguchi, S., Wing, K., Onishi, Y., Prieto-Martin, P. and Yamaguchi,

T., Regulatory T cells: how do they suppress immune responses? Int.

Immunol. 2009. 21: 1105–1111.

4 Costantino, C. M., Baecher-Allan, C. M. and Hafler, D. A., Human regula-

tory T cells and autoimmunity. Eur. J. Immunol. 2008. 38: 921–924.

5 Klareskog, L., Ronnelid, J. and Holm, G., Immunopathogenesis and

immunotherapy in rheumatoid arthritis: an area in transition. J. Intern.

Med. 1995. 238: 191–206.

6 Cao, D., Malmstrom, V., Baecher-Allan, C., Hafler, D., Klareskog, L.

and Trollmo, C., Isolation and functional characterization of regulatory

CD25brightCD4+ T cells from the target organ of patients with rheumatoid

arthritis. Eur. J. Immunol. 2003. 33: 215–223.

7 Cao, D., van Vollenhoven, R., Klareskog, L., Trollmo, C. and Malmstrom,

V., CD25brightCD4+ regulatory T cells are enriched in inflamed joints of

patients with chronic rheumatic disease. Arthritis Res. Ther. 2004. 6: R335–

R346.

8 van Amelsfort, J. M., Jacobs, K. M., Bijlsma, J. W., Lafeber, F. P. and

Taams, L. S., CD4(+)CD25(+) regulatory T cells in rheumatoid arthritis:

differences in the presence, phenotype, and function between peripheral

blood and synovial fluid. Arthritis Rheum. 2004. 50: 2775–2785.

9 Maliszewski, C. R., Delespesse, G. J., Schoenborn, M. A., Armitage, R. J.,

Fanslow, W. C., Nakajima, T., Baker, E. et al., The CD39 lymphoid cell

activation antigen. Molecular cloning and structural characterization. J.

Immunol. 1994. 153: 3574–3583.

10 Koziak, K., Sevigny, J., Robson, S. C., Siegel, J. B. and Kaczmarek, E., Anal-

ysis of CD39/ATP diphosphohydrolase (ATPDase) expression in endothe-

lial cells, platelets and leukocytes. Thromb. Haemost. 1999. 82: 1538–1544.

11 Deaglio, S., Dwyer, K. M., Gao, W., Friedman, D., Usheva, A., Erat, A.,

Chen, J. F. et al., Adenosine generation catalyzed by CD39 and CD73

expressed on regulatory T cells mediates immune suppression. J. Exp.

Med. 2007. 204: 1257–1265.

12 Kaczmarek, E., Koziak, K., Sevigny, J., Siegel, J. B., Anrather, J., Beau-

doin, A. R., Bach, F. H. et al., Identification and characterization of

CD39/vascular ATP diphosphohydrolase. J. Biol. Chem. 1996. 271: 33116–

33122.

13 Dwyer, K. M., Deaglio, S., Gao, W., Friedman, D., Strom, T. B. and Robson,

S. C., CD39 and control of cellular immune responses. Purinergic Signal.

2007. 3: 171–180.

14 Huang, S., Apasov, S., Koshiba, M. and Sitkovsky, M., Role of A2a extra-

cellular adenosine receptor-mediated signaling in adenosine-mediated



inhibition of T-cell activation and expansion. Blood 1997. 90:

1600–1610.

15 Fletcher, J. M., Lonergan, R., Costelloe, L., Kinsella, K., Moran, B.,

O’Farrelly, C., Tubridy, N. et al., CD39+Foxp3+ regulatory T cells sup-

press pathogenic Th17 cells and are impaired in multiple sclerosis. J.

Immunol. 2009. 183: 7602–7610.

16 Moncrieffe, H., Nistala, K., Kamhieh, Y., Evans, J., Eddaoudi, A., Eaton,

S. and Wedderburn, L. R., High expression of the ectonucleotidase CD39

on T cells from the inflamed site identifies two distinct populations,

one regulatory and one memory T cell population. J. Immunol. 2010. 185:

134–143.

17 Borsellino, G., Kleinewietfeld, M., Di Mitri, D., Sternjak, A., Diamantini,

A., Giometto, R., Hopner, S. et al., Expression of ectonucleotidase CD39

by Foxp3+ Treg cells: hydrolysis of extracellular ATP and immune sup-

pression. Blood 2007. 110: 1225–1232.

18 Herrath, J., Muller, M., Amoudruz, P., Janson, P., Michaelsson, J., Lars-

son, P. T., Trollmo, C. et al., The inflammatory milieu in the rheumatic

joint reduces regulatory T-cell function. Eur. J. Immunol. 2011. 41:

2279–2290.

19 de Kleer, I. M., Wedderburn, L. R., Taams, L. S., Patel, A., Varsani,

H., Klein, M., de Jager, W. et al., CD4+CD25bright regulatory T cells

actively regulate inflammation in the joints of patients with the

remitting form of juvenile idiopathic arthritis. J. Immunol. 2004. 172:

6435–6443.

20 Feger, U., Luther, C., Poeschel, S., Melms, A., Tolosa, E. and Wiendl,

H., Increased frequency of CD4+ CD25+ regulatory T cells in the cere-

brospinal fluid but not in the blood of multiple sclerosis patients. Clin.

Exp. Immunol. 2007. 147: 412–418.

21 Holmen, N., Lundgren, A., Lundin, S., Bergin, A. M., Rudin, A., Sjo-

vall, H. and Ohman, L., Functional CD4+CD25high regulatory T cells are

enriched in the colonic mucosa of patients with active ulcerative colitis

and increase with disease activity. Inflamm. Bowel Dis. 2006. 12: 447–456.

22 Korn, T., Reddy, J., Gao, W., Bettelli, E., Awasthi, A., Petersen, T. R.,

Backstrom, B. T. et al., Myelin-specific regulatory T cells accumulate in

the CNS but fail to control autoimmune inflammation. Nat. Med. 2007.

13: 423–431.

23 Perry, C., Hazan-Halevy, I., Kay, S., Cipok, M., Grisaru, D., Deutsch, V.,

Polliack, A. et al., Increased CD39 expression on CD4(+) T lymphocytes

has clinical and prognostic significance in chronic lymphocytic leukemia.

Ann. Hematol. 2012. 91: 1271–1279.

24 Mandapathil, M., Szczepanski, M. J., Szajnik, M., Ren, J., Lenzner, D. E.,

Jackson, E. K., Gorelik, E. et al., Increased ectonucleotidase expression

and activity in regulatory T cells of patients with head and neck cancer.

Clin. Cancer Res. 2009. 15: 6348–6357.

25 Kas-Deelen, A. M., Bakker, W. W., Olinga, P., Visser, J., de Maar, E. F.,

van Son, W. J., The, T. H. et al., Cytomegalovirus infection increases the

expression and activity of ecto-ATPase (CD39) and ecto-5’nucleotidase

(CD73) on endothelial cells. FEBS Lett. 2001. 491: 21–25.

26 Leal, D. B., Streher, C. A., de M. Bertoncheli, C., Carli, L. F., Leal, C.

A., da Silva, J. E., Morsch, V. M. et al., HIV infection is associated with

increased NTPDase activity that correlates with CD39-positive lympho-

cytes. Biochim. Biophys. Acta 2005. 1746: 129–134.

27 Regateiro, F. S., Cobbold, S. P. and Waldmann, H., CD73 and adenosine

generation in the creation of regulatory microenvironments. Clin. Exp.

Immunol. 2013. 171: 1–7.

28 Eltzschig, H. K., Ibla, J. C., Furuta, G. T., Leonard, M. O., Jacobson, K. A.,

Enjyoji, K., Robson, S. C. et al., Coordinated adenine nucleotide phospho-

hydrolysis and nucleoside signaling in posthypoxic endothelium: role of

ectonucleotidases and adenosine A2B receptors. J. Exp. Med. 2003. 198:

783–796.

29 Nakamachi, Y., Koshiba, M., Nakazawa, T., Hatachi, S., Saura, R.,

Kurosaka, M., Kusaka, H. et al., Specific increase in enzymatic activity

of adenosine deaminase 1 in rheumatoid synovial fibroblasts. Arthritis

Rheum. 2003. 48: 668–674.

30 Zhou, Q., Yan, J., Putheti, P., Wu, Y., Sun, X., Toxavidis, V., Tigges, J.

et al., Isolated CD39 expression on CD4+ T cells denotes both regulatory

and memory populations. Am. J. Transplant. 2009. 9: 2303–2311.

31 Dwyer, K. M., Hanidziar, D., Putheti, P., Hill, P. A., Pommey, S., McRae,

J. L., Winterhalter, A. et al., Expression of CD39 by human peripheral

blood CD4+ CD25+ T cells denotes a regulatory memory phenotype. Am.

J. Transplant. 2010. 10: 2410–2420.

32 Melton, A. C., Melrose, J., Alajoki, L., Privat, S., Cho, H., Brown, N., Plavec,

A. M. et al., Regulation of IL-17A production is distinct from IL-17F in a

primary human cell co-culture model of T cell-mediated B cell activation.

PLoS One 2013. 8: e58966.

33 Adamik, J., Henkel, M., Ray, A., Auron, P. E., Duerr, R. and Barrie, A.,

The IL17A and IL17F loci have divergent histone modifications and are

differentially regulated by prostaglandin E2 in Th17 cells. Cytokine 2013.

64: 404–412.

34 Stummvoll, G. H., DiPaolo, R. J., Huter, E. N., Davidson, T. S., Glass, D.,

Ward, J. M. and Shevach, E. M., Th1, Th2, and Th17 effector T cell-induced

autoimmune gastritis differs in pathological pattern and in susceptibility

to suppression by regulatory T cells. J. Immunol. 2008. 181: 1908–1916.

35 Lubberts, E., Koenders, M. I., Oppers-Walgreen, B., van den Bersselaar,

L., Coenen-de, Roo, C. J., Joosten, L. A. and van den Berg, W. B., Treat-

ment with a neutralizing anti-murine interleukin-17 antibody after the

onset of collagen-induced arthritis reduces joint inflammation, cartilage

destruction, and bone erosion. Arthritis Rheum. 2004. 50: 650–659.

36 Lister, M. F., Sharkey, J., Sawatzky, D. A., Hodgkiss, J. P., Davidson, D. J.,

Rossi, A. G. and Finlayson, K., The role of the purinergic P2×7 receptor

in inflammation. J. Inflamm. (Lond.) 2007. 4: 5.

37 Patel, D. D., Lee, D. M., Kolbinger, F. and Antoni, C., Effect of IL-17A

blockade with secukinumab in autoimmune diseases. Ann. Rheum. Dis.

2013. 72(Suppl 2): ii116–ii123.

38 Burgess, A., Vigneron, S., Brioudes, E., Labbe, J. C., Lorca, T. and Castro,

A., Loss of human Greatwall results in G2 arrest and multiple mitotic

defects due to deregulation of the cyclin B-Cdc2/PP2A balance. Proc. Natl.

Acad. Sci. USA 2010. 107: 12564–12569.

39 Baron, U., Floess, S., Wieczorek, G., Baumann, K., Grutzkau, A., Dong, J.,

Thiel, A. et al., DNA demethylation in the human FOXP3 locus discrim-

inates regulatory T cells from activated FOXP3(+) conventional T cells.

Eur. J. Immunol. 2007. 37: 2378–2389.

Abbreviations: HC: healthy control · JIA: juvenile idiopathic arthritis ·PB: peripheral blood · RA: rheumatoid arthritis · SF: synovial fluid · SpA:

ankylosing spondylitis · Teff: T effector · TSDR: Treg-specific demethy-

lated region

Full correspondence: Dr. Vivianne Malmstrom, Rheumatology ResearchUnit, CMM L8:04, Karolinska University Hospital, 171 76 Stockholm,SwedenFax: +46-8-5177-5562e-mail: [email protected]

Received: 3/10/2013Revised: 16/6/2014Accepted: 30/6/2014Accepted article online: 2/7/2014


surface expression of cd39 identifies an enriched treg-cell subset in the rheumatic joint, which...

Documents