Generation and Accumulation of Immunosuppressive Adenosine by Human CD4⁺CD25^highFOXP3⁺ Regulatory T Cells^*

Healthy Volunteers

Peripheral venous blood samples for phenotypic and functional analyses of lymphocyte subsets were obtained from 15 normal controls. All subjects signed an informed consent approved by the Institutional Review Board of the University of Pittsburgh. The normal control group included 4 males and 11 females with a mean age of 39 years (range, 24–58 years).

Collection of PBMCs

Blood samples (20–30 ml) were drawn into heparinized tubes and centrifuged on Ficoll-Hypaque gradients (Amersham Biosciences). PBMCs were recovered, washed in AIM-V medium (Invitrogen), counted in a trypan blue dye, and immediately used for experiments evaluating the phenotype.

Separation of Treg

CD4⁺CD25^neg as well as CD4⁺CD25⁺ T cells were freshly isolated from buffy coats using the Regulatory T cell Separation Kit and AutoMACS (Miltenyi Biotech). After the cells were washed twice, the CD4⁺ T cells were negatively selected first from the total PBMCs and, afterward, positive selection was performed on anti-CD25 magnetic beads, separating CD4⁺CD25^neg cells and CD4⁺CD25⁺ cells. The purity of each cell fraction was >97%. CD4⁺CD25^high cells (i.e. those with an MFI of >120) were single cell-sorted following staining with an anti-CD25 antibody and using a Beckman Coulter cell sorter.

Antibodies

The following anti-human monoclonal antibodies were used for flow cytometry: anti-CD3-ECD, anti-CD4-ECD, anti-CD4-PC5, anti-CD8-PC5, anti-CD25-PC5, anti-GITR-FITC, anti-FOXP3-FITC, anti-CD39-FITC, anti-CD39-PE, anti-CD26-PE, anti-CD73-PE, unconjugated anti-CD73, and anti-CTLA4-PE. Antibodies and their respective isotypes, which served as a negative control for surface as well as intracellular staining, were purchased from Beckman Coulter, except for anti-FOXP3, anti-CD39-FITC, and anti-CD39-PE, which were purchased from eBioscience; anti-CD26-PE and anti-CD73-PE were purchased from BD Pharmingen; anti-GITR-FITC and anti-CTLA4-PE were purchased from R&D Systems; unconjugated anti-CD73 (clone: 10f1) was purchased from Santa Cruz Biotechnology; and FITC-conjugated AffiniPure Goat anti-mouse secondary antibody was purchased from Jackson ImmunoResearch. Before use, all antibodies were titrated using resting as well activated PBMCs obtained from normal controls to determine the optimal staining dilution for each antibody.

Surface and Intracellular Staining

Freshly isolated cells or activated cells were stained for flow cytometry as previously described (37). Briefly, cells were incubated with the antibodies for surface markers for 30 min at 4 °C in the dark and then fixed with 2% (w/v) paraformaldehyde in PBS for 15 min. Afterward, cells were permeabilized with 0.1% (w/v) saponin and stained with antibodies specific for intracellular markers for 30 min at 4 °C in the dark. Cells were washed twice with 0.1% saponin in PBS, resuspended in a flow solution and immediately analyzed by flow cytometry. Appropriate isotype controls were included for each sample.

Flow Cytometry

Flow cytometry was performed using an EPICS® XL-MCL flow cytometer equipped with Expo32 software (Beckman Coulter). The acquisition and analysis gates were restricted to the lymphocyte gate based on characteristic properties of the cells in the forward (FSC) and side scatter (SSC). Forward and side scatter were set in a linear scale, and 10⁵ cells were acquired for analysis, which was performed using the Coulter EXPO 32vl.2 analysis program. For additional analyses, gates were restricted to the CD3⁺CD4⁺, CD3⁺CD8⁺, or different CD4⁺CD25⁺ T-cell subsets.

Immunostaining

Single cell-sorted CD4⁺CD25^high and CD4⁺CD25^neg cells were cytocentrifuged onto glass slides and stained using a standard immunoperoxidase method. Cells were first fixed using a 1:1 methanol/acetone solution and then dried at room temperature for 4 h. Afterward, cells were treated with a serum-free protein block (Dako) for 1 h at room temperature, followed by washing with PBS and an overnight incubation at 4 °C in the dark with the primary Ab. The following Abs were used: unconjugated anti-human CD73 antibody (1:100 dilution, Santa Cruz Biotechnology); rabbit anti-human CD26 Ab; mouse anti-human ADA or appropriate isotype controls. Slides were then washed and, for CD73 staining, incubated with a secondary peroxidase-conjugated goat anti-mouse antibody for 1 h (Dako). Finally, peroxidase activity was developed using a liquid diaminobenzidine chromogen system (DAB⁺, Dako). Sections were washed and counterstained with hematoxylin (Dako). Slides were mounted with Glycergel Mounting Medium (DakoCytomation) and analyzed under a light microscope. For CD26 and ADA staining, slides were incubated with the following secondary antibodies: donkey anti-rabbit-FITC (1:200, Santa Cruz Biotechnology) and donkey anti-mouse-Cy3 (1:500, Jackson ImmunoResearch). Afterward slides were washed, fixed, and evaluated in an inverted Olympus FluoView 1000 laser scanning confocal microscope under an oil immersion objective (Center for Biology Imaging Core Facility, University of Pittsburgh). For digital image analysis, the software Adobe Photoshop version 7.0 was use.

Suppression Assay

Single cell-sorted CD4⁺CD39⁺, CD4⁺CD26^neg or CD4⁺CD25^high, and CD4⁺CD39^neg or CD4⁺CD26⁺ cells were tested for suppressor activity in a co-culture with CD4⁺CD25^neg responder cells (RCs). To evaluate their proliferation, RCs separated by single cell sorting were stained with 1.5 μm CFSE (Molecular Probes/Invitrogen) and incubated at 37 °C for 15 min. The unbound CFSE was quenched out by using an equal amount of fetal calf serum (Invitrogen), and subsequently cells were washed twice with phosphate-buffered saline. CFSE-labeled autologous CD4⁺CD25^neg 10⁵ cells/well were incubated in wells of flat-bottom 96-well plates at the suppressor cell (S)/RC ratios of 1:1, 1:2, 1:5, and 1:10. Using the same assay design, various inhibitors or modifiers, which were initially titrated to obtain the optimal concentration, were added to RC 30 min before adding the S cells. These were as follows: ARL67165 (250 μm), adenosine 5′-(α,β-methylene)diphosphate (100 μm), MRS 1191 (0.5 μm), erythro-9-(2-hydroxy-3-nonyl)adenine (2 μm) all purchased from Sigma-Aldrich; ZM241385 (0.3 μm), MRS 1706 (0.5 μm), and dipropylcyclopentylxanthine (0.5 μm) was purchased from Tocris Bioscience. To induce proliferation, RC were stimulated with plate-bound OKT-3 (2 μg/ml) and soluble anti-CD28 mAb (2 μg/ml, Miltenyi) in the presence of 150 IU/ml IL-2 for 5 days. S cells were considered to mediate suppression, when they significantly inhibited proliferation of RC in co-culture assays. All CFSE data were analyzed using the ModFit software provided by Vertify Software House (Topsham). The percentage of suppression was calculated by using the mean proliferation index of RC alone compared with the proliferation index of cultures containing RC + S cells. The program determines the percentage of cells within each detected peak, whereas the sum of all peaks in the control culture is set to be 100% proliferation or 0% suppression (1).

ATP Hydrolysis Assay

MACS-sorted CD4⁺CD25^neg or CD4⁺CD25⁺ cells (25 × 10³/well) were incubated in wells of flat-bottom 96-well plates for 30 min with various concentrations of exogenous ATP (10 μm, 25 μm, and 50 μm, Sigma-Aldrich). Some wells were preincubated with ARL67156, an ectonucleotidase inhibitor, at the final concentration of 250 μm for 30 min prior to the addition of exogenous ATP. The concentration of “unhydrolyzed” ATP was determined by measuring the frequency of luminescent events (CPM) in a luciferase-based detection system (ATP Lite Luminescence ATP Detection Assay System from PerkinElmer). CPM were recalculated into adenosine produced in nanomoles/1 million cells. The plates were examined in a Microplate Scintillation and Luminescence Counter (Packard). The average count was determined from triplicate wells.

Mass Spectrometric Analysis of Adenosine Production

CD4⁺CD25^neg or CD4⁺CD25⁺ T cells (25,000 cells/well) were incubated with 10 μm exogenous ATP in wells of 96-well flat bottom plates. To some wells ARL67156 (250 μm) or α,β-methylene-ADP (100 μm) was added 30 min prior to the assay. Cell supernatants were collected after various periods of incubation time. Samples were boiled for 2 min and stored on dry ice until analysis. Adenosine was measured on a ThermoFinnigan LCQ Duo mass spectrometer equipped with electrospray ionization. The samples were separated using a C18 column (Eclipse XDB-C18, 4.6 × 150 mm, 5 μm). A mobile phase consisted of 0.1% formic acid water and methanol solution. The flow rate of a mobile phase was 0.6 ml/min. The analytes were monitored with single-ion monitoring in the positive-ion mode; for adenosine the mass-to-charge ratio was 268; for 10–13 C-adenosine (internal standard) the mass-to-charge ratio was 278. The internal standard (10–13 C-adenosine) in supernatants is 10 pg/μl. The average concentration of adenosine was determined in duplicate wells.

Western Blots

Whole cell protein extracts were prepared from CD4⁺CD25^neg as well as CD4⁺CD25^high T cells, which were freshly isolated from buffy coats as described above. Afterward, cells were washed twice and lysed at 4 °C in a lysis buffer containing 0.5% Nonidet P-40, 150 mm NaCl, and 50 mm Tris base (Sigma). Laemmli loading buffer (4% SDS, 10% β-mercaptoethanol 20% glycerol, 0.004% bromphenol blue, 0.125 m Tris-HCl) was added to the cell lysates at a 1:1 ratio (v/v), and the lysates were then boiled for 5 min. Protein extracts were subjected to electrophoresis on 4–15% Tris-HCl gradient gels (Bio-Rad) and were subsequently transferred to polyvinylidene fluoride membranes (Millipore). The membranes were treated with polyclonal primary antibodies specific for human CD39 (Santa Cruz Biotechnology, rabbit 1:200), CD73 (Abcam, mouse, 1:200), CD26 (Santa Cruz Biotechnology, rabbit, 1:200), and ADA (Santa Cruz Biotechnology, mouse, 1:100) followed by goat anti-rabbit or goat anti-mouse secondary antibody conjugated to horseradish peroxidase (Pierce, goat, 1:40,000). SuperSignal West Femto Maximum Sensitivity Substrate (Pierce) and Kodak BioMax MR Film were used to visualize the target proteins.

Statistical Analysis

All data were presented as the means of at least three experiments ± 1 S.D. The data were analyzed using the Student's t test, and the p values of <0.05 were considered to be significant. Correlations were calculated using the Spearman rank correlation test.

Expression of CD39, CD73, CD26, and ADA on Treg versus CD4⁺ T Effector Cells

Lymphocytes in PBMCs were identified based on their forward scatter (FS) and sideward scatter (SS) characteristics (Fig. 1A). Next, the frequency of CD4⁺CD25^high and CD4⁺CD25^neg cells in the total CD3⁺CD4⁺ cell population was determined. Gating strategies are shown in Fig. 1B. CD4⁺ cells with CD25 expression ≥120 MFI were considered as CD4⁺CD25^high cells; those with a MFI <120 as CD25^{intermediate/low} (1). The majority of the CD4⁺CD25^high cells were positive for FOXP3 (mean of 75.6%) and mediated strong suppression, as previously reported by us (1). Double staining revealed that the majority of CD3⁺CD4⁺ T cells were CD39^negCD26⁺ and 7.5% were CD39⁺CD26^neg. Only a very small portion of cells (0.2%) were CD39⁺CD26⁺ (Fig. 1C). These data shown in Fig. 1C confirm that CD39 and CD26 are expressed on different subsets of CD4⁺ T cells.

Next, CD39 expression on human CD4⁺CD25^high and CD4⁺CD25^neg cells was examined by flow cytometry (Fig. 1, D and E). As shown in Fig. 1E, only 8 ± 4% of cells within the CD3⁺CD4⁺ cell subset were CD39⁺. The CD4⁺CD25^high subset was highly enriched in CD39⁺ cells (79 ± 15%, p < 0.001) relative to CD4⁺CD25^neg cells (5 ± 2%). The CD4⁺CD25^{intermediate/low} cell subset contained T cells with low FOXP3 (11 ± 2%) and CD39 (9 ± 3%) expression but a relatively high CD26 surface expression (76 ± 7%). Neither the CD4⁺CD25^low subset (data not shown) nor the CD3⁺CD8⁺ subset (Fig. 1E) expressed CD39. These results indicate that CD39 expression is largely restricted to CD4⁺CD25^high Treg. In contrast, CD26 was broadly expressed on >80% of all CD3⁺CD4⁺ cells (Fig. 1E). Gating on CD4⁺CD25^high and CD4⁺CD25^neg T cell subsets, we observed a significantly higher CD26 expression on CD4⁺CD25^neg subset relative to CD4⁺CD25^high Treg (Fig. 1, D and E), suggesting that CD26 was preferentially expressed on the conventional CD4⁺ T cells. Its expression increased significantly upon T-cell activation (data not shown).

CD73 surface expression was very low in the CD4⁺CD25^high T-cell subset, ranging from below 1 up to 7% positive cells (Fig. 2A). This is in contrast to murine Treg, which were reported to express high levels of cell membrane-associated CD73 (21). Although no difference in the level of CD7 expression on the cell surface was seen between CD4⁺CD25^high versus CD4⁺CD25^neg T cell subsets, intracellular staining using permeabilized Treg followed by flow cytometry showed that most CD4⁺CD25^high Treg (71 ± 5%) were positive for CD73 (Fig. 2A). In addition, mean fluorescence intensity (MFI) measurements of CD73 expression levels in permeabilized T cells showed that the CD4⁺CD25^high cells had higher levels of CD73 expression (p < 0.001) than CD4⁺CD25^neg T cells (Fig. 2B).

Given the observed differential expression of ectonucleotidases and CD26 in the T cell subsets, we next performed Western blot analyses using freshly isolated and single cell-sorted CD4⁺CD25^high and CD4⁺CD25^neg cells. Because CD26 expression was previously reported to be associated with ADA (25), we also determined the levels of ADA in both these cell subsets. As expected, CD39 and CD73 were expressed in CD4⁺CD25^high Treg but not in CD4⁺CD25^neg conventional CD4⁺ T cells (Fig. 2C). In contrast, CD26 and ADA were predominantly expressed in CD4⁺CD25^neg T cells and only weakly in Treg (Fig. 2C). To further confirm that both CD26 and ADA expression levels were high in CD4⁺CD25^neg conventional T cells and low in Treg, confocal microscopy was performed using single cell-sorted cells. Fig. 2D shows high levels of both markers on the surface of CD25^neg conventional CD4⁺ T cells and low expression levels in CD4⁺CD25^high Treg. It also convincingly demonstrates co-expression of CD26 and ADA on the surface of conventional CD4⁺ T cells (Fig. 2D).

Phenotypic Characterization of the CD4⁺CD39⁺ and CD4⁺CD26^neg T-cell Fractions

Our data showed that CD39 and CD26 potentially define distinct subsets of CD4⁺ T cells. To further characterize the Treg subpopulations that expressed CD39 versus conventional CD4⁺ T cells with high CD26 expression, additional multiparameter flow cytometry analyses were performed. Gating on CD4⁺CD25^high cells, we again observed enrichment in CD39⁺ cells and low percentages of CD26⁺ and GITR⁺ cells (Fig. 3A). When gates were set on CD39⁺ or CD26^neg subsets of CD4⁺ cells, a similar marker profile was observed (Fig. 3, B and C), suggesting a reciprocal relationship between these subsets. As shown in Fig. 3D, CD25^high expression levels correlated negatively with those for CD26 on CD4⁺ T cells (r = 0.56). Within the CD4⁺CD25^high Treg subset, levels of CD39 expression correlated negatively with those for CD26 (r = 0.836) (Fig. 3E), whereas a positive correlation was seen for the levels of FOXP3 and CD39 expression in CD4⁺CD25^high cells (r = 0.90) (Fig. 3F). These results confirm that CD39 is a biomarker of CD4⁺FOXP3⁺ and CD4⁺CD25^high Treg.

Suppressor Function of CD4⁺CD39⁺ and CD4⁺CD26^neg T-cell Subsets

To analyze suppressor activity of CD4⁺CD39⁺ and CD4⁺CD26^neg cells, these cell subsets were single cell sorted from freshly isolated PBMCs. The sorted cells serving as suppressor(s) were co-incubated with autologous CD4⁺CD25^neg RC at different S/RC ratios. After a 5-day culture, the mean suppressor activity of CD4⁺CD39⁺ cells at the 1S:1RC ratio was 43 ± 3%, and the suppression of proliferation linearly decreased upon further dilution of S (Fig. 4A). The mean suppressor activity of CD4⁺CD26^neg cells at the 1S:1RC ratio was 24 ± 1%, and it also significantly decreased upon S cell dilution (Fig. 4B). CD4⁺CD39^neg as well as CD4⁺CD26⁺ cells did not suppress proliferation of RCs. These results indicate that both CD39⁺ and CD26^neg subsets of Treg mediate suppressive activity in conventional CFSE-based proliferation assays with autologous RC. The somewhat lower suppressive effects of CD26^neg cells relative to CD39⁺ cells can be explained by the fact that not all CD26^neg cells expressed CD39 (Fig. 3, B and C).

Hydrolysis of ATP by CD4⁺CD25⁺ versus CD4⁺CD25^neg Cells

Because CD39 is an ectonucleotidase hydrolyzing ATP, it was important to determine whether the isolated Treg expressing this marker also had enzymatic activity. Single cell-sorted CD4⁺CD25⁺ cells and CD4⁺CD25^neg cells were stimulated with OKT-3 and anti-CD28 Ab for 3 days and afterward incubated with various concentrations of exogenous ATP for 30 min. In these and subsequent analyses of T-cell functions, we used CD4⁺CD25⁺ and not CD4⁺CD25^high T cells because of a requirement for relatively high numbers of Treg in these functional assays. Compared with autologous CD4⁺CD25^neg cells, CD4⁺CD25⁺ Treg hydrolyzed significantly more ATP (Fig. 5A; p < 0.02 to 0.001). Upon pretreatment with ARL67156, a selective inhibitor of ecto-ATPases, the capability of CD4⁺CD25⁺ cells to hydrolyze ATP was decreased at different ATP concentrations (Fig. 5B). Whereas the inhibitors did not show any effect on CD4⁺CD25^neg cells (data not shown). We also noted that unstimulated, freshly isolated CD4⁺CD25⁺ cells hydrolyzed the same amount of exogenous ATP as stimulated CD4⁺CD25⁺ cells, but needed more time for hydrolysis (data not shown).

Adenosine Generation by CD4⁺CD25⁺ Cells versus CD4⁺CD25^neg Cells

To further analyze the activity of the ectonucleotidases CD39 and CD73 in Treg, we determined the ability of activated CD4⁺CD25⁺ and CD4⁺CD25^neg cells to produce adenosine following the addition of 10 μm exogenous ATP. Adenosine levels were measured in the cell supernatants collected at various time points after ATP addition. As shown in Fig. 5C, CD4⁺CD25⁺ cells produced significantly more adenosine than CD4⁺CD25^neg cells. Upon co-incubation of CD4⁺CD25⁺ cells with ARL67156, adenosine production was almost completely blocked (p < 0.09). Also, we observed a complete inhibition of adenosine production by CD4⁺CD25⁺ as cells when α,β-methylene ADP, a specific CD73 inhibitor, was added to selected wells (Fig. 5D). Both inhibitors did not have any effects on CD4⁺CD25^neg cells (data not shown). Next, using the same experimental design, we incubated both cell types with 1 μm exogenous adenosine and harvested the cell supernatant after various time points. In Treg cultures, the amount of unmetabolized adenosine was four times higher than in CD4⁺CD25^neg cell cultures (data not shown).

Exogenous Adenosine Suppresses Proliferation of CD4⁺CD25^neg T Cells

To show that exogenous adenosine suppresses proliferation of CD4⁺CD25^neg RCs, we incubated these freshly isolated CFSE-labeled cells with various concentrations of 2-chloroadenosine for 5 days. Fig. 6A shows a dose-dependent suppression of RC proliferation in the presence of 2-chloroadenosine, used here as an analog of exogenous adenosine.

Effects of Ectonucleotidase and ADA Inhibitors on Suppression Mediated by CD4⁺CD39⁺ Cells

As CD4⁺CD39⁺ T cells mediated suppression of RC proliferation (Fig. 6B), we next used these cells isolated by AutoMACs to confirm that adenosine produced during ATP cleavage by the associated ectonucleotidases is responsible for suppression of RCs. We showed that ARL67156, a structural analogue of ATP and an ectonucleotidase inhibitor, added to co-cultures of CD4⁺CD39⁺ cells serving as S cells with autologous RCs significantly decreased suppression levels (p < 0.02) compared with cultures without the inhibitor (38 ± 9% versus 19 ± 1%) (Fig. 6B). In the same type of co-culture, the addition of α,β-methylene-ADP, an inhibitor of CD73, also significantly reduced (p < 0.03) Treg-mediated suppression (Fig. 6C). In the presence of erythro-9-(2-hydroxy-3-nonyl)adenine, an inhibitor of ADA, suppression mediated by Treg was significantly increased (p < 0.02) compared with cultures without the inhibitor (36 ± 1 versus 52 ± 4%) (Fig. 6C). Thus, blocking of ADA activity in the co-cultures resulted in a higher level of adenosine produced by Treg and greater Treg-mediated suppression of RC proliferation.

Effects of an Adenosine Receptor Antagonist on Suppression Mediated by CD4⁺CD39⁺ Cells

The immunosuppressive effects of adenosine are mediated via the A_2aR, which is expressed on effector T cells (1). We hypothesized that, if adenosine suppresses proliferation of RCs, then blocking of the A_2aR with a specific antagonist should inhibit Treg-mediated suppression. To test this hypothesis, we added ZM241385, a selective A_2a and A_2b receptor antagonist, to the above described S/RC co-cultures. The addition of ZM241385 almost completely blocked the suppression mediated by CD4⁺CD39⁺ cells at the 1S:1RC ratio (9 ± 1% versus 36 ± 1%; p < 0.001) (Fig. 6D). The addition of dipropylcyclopentylxanthine, a selective A1 receptor antagonist, or MRS1191, a selective A₃ receptor antagonist, or MRS1706, a selective A_2b receptor antagonist, did not show any effect on CD39⁺ T cell-mediated suppression (data not shown). All antagonists incubated with RCs in the absence of S cells did not influence the proliferation of RCs.