Mesenchymal stromal cells (MSCs)
The MSCs used in this study were obtained from Normal cervix (NCx) tissues, that were obtained from three subjects who had hysterectomy surgery not related to cancer, and CeCa samples were obtained from biopsies from three patients in stage IIIB. The local ethics committee approved these procedures. The CeCa samples intended for tissue culture were collected from biopsies sent to the Pathology Department for routine diagnosis. NCx- and CeCa-derived mesenchymal stromal cells (MSCs) were obtained by enzymatic digestion as previously described , and maintained in low glucose Dulbecco’s Modified Eagle Medium (DMEM; Gibco Laboratories, Grand Island, NY, USA) supplemented with 15 % fetal bovine serum (FBS; Gibco), 100 IU/ml penicillin, 100 μg/ml streptomycin (Gibco) and 5 mM l-glutamine at 37 °C with 5 % CO2.
MSCs were characterized based on the morphological, phenotypic and differentiation parameters performed according to protocols previously reported by our working group . FITC, PE or APC-conjugated monoclonal antibodies against CD73, CD90 and CD45 (BD Biosciences, San Diego, CA, USA) CD105, CD13, CD14, (Caltag, Buckingham, UK), HLA-ABC, HLA-DR, CD31 and CD34 (Invitrogen, Carlsbad, CA, USA) were used for immunophenotypic characterizations and analyzed on a CyAN cytometer (Beckman Coulter, Fullerton CA, USA). Adipogenic and osteogenic differentiation was induced with Stem Cells Kits™ (STEMCELL Technologies, Inc., Vancouver, BC, Canada). Adipogenic differentiation was determined by visualizing the presence of Oil Red O-stained (Sigma-Aldrich, St. Louis, MO, USA) lipid vacuoles. Osteogenic differentiation was assessed by alkaline phosphatase staining. Chondrogenic differentiation was induced with a commercial induction medium (Cambrex Bio Science, Walkersville, Inc., Maryland, USA) that was supplemented with 10 ng/ml of TGF-β (Cambrex). The resulting micromasses were fixed, embedded and sliced. Cross-sections were stained with Alcian blue dye (Sigma-Aldrich).
Expression of CD39 and CD73 in MSCs
The expression of the ectonucleotidases CD39 and CD73 in MSC cell membranes was determined by labeling with anti-CD39-FITC (eBioscience, San Diego, CA, USA) and anti-CD73-PE (BD Bioscience) monoclonal antibodies according to the manufacturers’ instructions. Cell analysis was performed from the acquisition of 25,000 events in a CyAN cytometer (Beckman Coulter, Fullerton CA, USA). In addition, the expression of the ectonucleotidases CD39 and CD73 in MSCs cell membranes was also determined by immunocytochemical staining by using human anti-CD39 and anti-CD73 mAbs (Novus, Cambridge, UK). Briefly, MSCs grown on glass coverslips to semiconfluence, were fixed in 2 % paraformaldehyde (Sigma, St. Louis, MO) in PBS for 6 min and permeabilized with 0.01 % Triton X-100 in PBS. Following incubation with 2 % (w/v) bovine serum albumin (Sigma, St. Louis, MO), cells were incubated for 1 h with the mAbs. Following washing, the coverslips were incubated for 1 h with a secondary antibody horseradish peroxidase-conjugated goat anti-mouse (DAKO, Carpinteria, USA). The development was performed with substrate-chromogen solution 3,3′-diaminobenzidine dihydrochloride (DAB) for 3–5 min. Secondary antibody alone was included as control for the experiments. The nuclei were stained with Mayer’s Hematoxylin. Glass coverslips were scanned to obtain electronic files. The immunocytochemical stains of two stains made independently were digitally analyzed using the Aperio CS (San Diego, CA, USA) digital pathology equipment.
Hydrolytic activity of CD39 and CD73
Samples containing 105 MSCs were cultured in presence of 5 mM ATP, ADP or AMP in 200 µl of low glucose DMEM supplemented with 10 % FBS to determine the enzymatic activity of the CD39 and CD73 ectonucleotidases. FBS was previously dialyzed with a membrane (molecular weight cutoff of 12 kDa). To inhibit the enzymatic activity of CD39 and CD73, MSCs were incubated in the presence of 5 mM specific inhibitors, including sodium polyoxotungstate (POM-1, Sigma-Aldrich, St Louis, MO, USA) and adenosine 5′-(α,β-methylene) diphosphate (APCP, Sigma-Aldrich). After 30 min of incubation with each inhibitor, the adenine nucleotides ATP, ADP and AMP were added to a final concentration of 5 mM. The total culture volumes were 200 µl. Supernatant samples were collected every 60 min, and the presence of Ado was analyzed by thin layer chromatography (TLC) and ultra-performance liquid chromatography (UPLC) (UPLC aquity, Waters, Milford MA, USA). To analyze the samples by TLC, 1 µl of each supernatant was loaded on fluorescent plates precoated with gel (Whatman, GE Healthcare, Freiburg, Germany). Samples were eluted for 1 h using a mobile phase a mixture composed of isobutanol:isoamyl alcohol:ethoxyethanol:ammonia:water in the ratio 9:6:18:9:15 [38, 39]. ATP, ADP, AMP, Ado and inosine (Sigma-Aldrich) at 5 mg/ml were used as standards. Finally, the compounds were visualized and photographed under an UV transilluminator (UVP Biodo-H System, Upland, CA, USA).
Ultra-performance liquid chromatography (UPLC)
An UPLC system (UPLC acquity, Waters) was used to quantify the amount of Ado generated in MSCs cultures in the presence of each nucleotide. Quantitative analysis of samples using standard quantities of synthetic Ado was carried out with Empower 3 software (Waters, USA). The mobile phase consisted of 0.5 % acetonitrile, 5 % methanol, and 94.5 % sodium acetate buffer 0.25 M, pH 6.3. Supernatant samples were centrifuged at 13,000 rpm and filtered on Amicon membranes with a cutoff of 3000 Da and subsequently diluted 1:200 with the mobile phase mixture. Run conditions were as follows: flow rate of 1.0 ml/min, UV detection at 254–260 nm, 2.0 min of retention time, room temperature, and a LiChrosfer 5 µm RP-18e 100 A (size 125 mm × 4 mm, 5 µm particle size) reverse phase column. Ado was quantified by comparing the retention time of the sample with that of the synthetic Ado used as standard.
In vitro suppression assay for testing T cell proliferation
To determine the suppressive activity of Ado on T-cell proliferation, 1 × 105 CD8+ T-cells isolated from peripheral blood mononuclear cells (PBMC) from normal donors by negative selection (EasySep Enrichment Cocktail, Stem Cell Technologies, Vancouver, BC, Canada) were cultured in triplicate in 96-well flat bottom plates (Corning, NY, USA) in 100 µl Iscove’s Modified Dulbecco’s Medium (IMDM) (Gibco) without phenol red, supplemented with 10 % dialyzed FBS. Beads coated with anti-CD2/CD3/CD28 antibodies (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany) were added to T-cell cultures in a 2:1 ratio of T-cells:beads. To determine the suppressive capacity of Ado contained in the supernatants from MSCs cultured in the presence of nucleotides, 20 µl of each supernatant was added to the T-cell cultures (total volume of 200 µl) and then incubated at 37 °C and 5 % CO2 for 72 h. Caffeine, a nonselective antagonist of the Ado receptor, was added to some culture plates at a concentration of 300 μM (J.T. Baker, Center Valley, PA, USA). In other cases, 1 µM ZM241385, a selective antagonist of A2A receptor, or ZM241385 (1 μM) plus caffeine (300 μM) were added. Lymphocytes stimulated with beads coated with anti-CD2/CD3/CD28 antibodies alone or in the presence of synthetic Ado (500 μM), caffeine (300 μM), Ado:caffeine (500 μM:300 μM), Ado:ZM241385 (500 μM:1 μM), Ado: ZM241385:caffeine (500 μM:1 μM:300 μM) or without stimulation were seeded independently to establish appropriate controls. The CellTiter 96® AQueous One Solution (Promega, Madison, WI, USA) commercial kit was used to determine T-cell proliferation according to the procedure provided in the supplier insert. After incubating the cells for 4 h at 37 °C and 5 % CO2, the plate was read on an ELISA plate reader (Corning) at a wavelength of 490 nm. The percentage of T-cell proliferation was determined as follows: % proliferation = [reading T-cell cultures with conditioned media and anti-CD2/CD3/CD28 beads]/[reading T-cell cultures with anti-CD2/CD3/CD28 beads] × 100.
Determination of IFN-γ in activated T-cells
A total of 2.5 × 105 CD8+ T-cells were cultured for 48 h in a 24-well plate (Corning) with 1 ml of IMDM culture medium + 10 % dialyzed FBS in the presence of beads coated with anti-CD2/CD3/CD28 in the ratio 2:1 and in the presence or absence of 20 μL of MSCs supernatants. Caffeine (300 μM) or ZM241385 (1 μM) were added to some wells. Lymphocytes stimulated with beads coated with anti-CD2/CD3/CD28 alone or in the presence of synthetic Ado (500 μM), caffeine (300 μM), Ado:caffeine (500 μM:300 μM), Ado: ZM241385 (500 mM:1 mM), Ado: ZM241385:caffeine (500 μM:1 μM:300 μM) or without stimulus were seeded independently to establish appropriate controls. During the last 4 h of culture, Brefeldin-A (Sigma-Aldrich) was added to a final concentration of 10 μM to determine the content of intracellular IFN-γ. Subsequently, cells were collected, fixed and permeated using the Cytofix/Cytoperm Kit (BD Biosciences, San Jose, CA, USA) kit. T-cells were labeled with anti-IFN-γ/FITC and anti-CD8/APC (R&D Systems, Inc, Minneapolis, MN, USA) monoclonal antibodies, incubated for 30 min at 4 °C and washed three times with PBS. The labeled cells were evaluated in a CyAN flow cytometer (Beckman Coulter). A total of 5 × 104 events were acquired to analyze the percentage of CD8+IFN-γ+ cells using Summit V4.3 software.
Measurements of cAMP levels were performed, as described previously . Briefly, CD8+ T-cells (4 × 105) were cultured alone or in the presence of 20 µl of each supernatant (total volume of 200 µl). In other cases, synthetic Ado (500 μM), caffeine (300 μM), Ado:caffeine (500 μM:300 μM), Ado: ZM241385 (500 mM:1 mM), Ado: ZM241385:caffeine (500 μM:1 μM:300 μM), or the ARs agonist, 5′-N-ethylcarboxamidoadenosine (NECA; Sigma-Aldrich) (5 μM) were seeded independently to establish appropriate controls. The cells were incubated for 30 min at 37 °C, and the reaction was stopped by addition of 1 N hydrochloric acid. cAMP levels were determined by ELISA using the Parameter™ cAMP assay kit (R&D Systems, Inc, Minneapolis, MN, USA).
In vitro analysis of the suppression of effector CTL activity
To analyze the suppressor capacity of Ado on effector CTL function, CD8+ T-cells specific for the YMLDLQPETT peptide derived from the E7 HPV-16 protein were obtained using a method previously reported by our group . A total of 1 × 106 CTLs were previously cultured for 3 h in the presence of synthetic Ado (500 µM) or with 20 µl of supernatants obtained from MSCs cultured for 5 h in presence of 5 mM AMP. Subsequently, CTLs were washed and challenged with cells from the T2 lymphoblastic cell line that express empty HLA-A*02:01 molecules on their cell surfaces , which were loaded with 10 µM YMLDLQPETT peptide.
Target cells (peptide-loaded T2) were labeled with 5(6)-carboxyfluorescein diacetate N-succinimidyl ester (CFSE) (Sigma-Aldrich), and CTLs were labeled with CD8-APC antibody (BD Bioscience) following the protocol provided by BD Bioscience. The cytotoxic activity of effector cells on target cells was determined in the proportions 10:1, 5:1 and 2.5:1 (effector:target) using a cell viability solution (7AAD, Sigma-Aldrich). Viable cells were analyzed under a Coulter CyAN flow cytometer (Beckman Coulter) from the acquisition of 100,000 events by determining the percentage of positive target cells for CFSE/7AAD.
Lymphocytes were independently seeded with medium alone or medium with AMP, synthetic Ado, caffeine (300 μM), ZM241385 (1 μM), Ado:caffeine (500 μM:300 μM), Ado:ZM241385 (500 mM:1 mM) or Ado: ZM241385:caffeine (500 μM:1 μM:300 μM) to establish the appropriate controls. A total of 5 % hydrogen peroxide was used in some cases to determine the total lysis of target cells.
The percentage of lysis was calculated according to the following formula: % cytotoxicity = 100 × [(experimental lysis (CFSE+, 7AAD+) − basal lysis (CFSE+, 7AAD+)/(total lysis (CFSE+, 7AAD+) − basal lysis (CFSE+, 7AAD+)].
All numerical data are presented as the mean value ± SEM of three independent experiments. Comparisons were evaluated by multivariate statistical analysis using GraphPad Prism version 7 (GraphPad Prism software, USA). The difference was considered statistically significant at P < 0.05.