Cell culture
Human peripheral blood mononuclear cells (PBMCs) were obtained from the New York Blood Center as leukopacks, following Institutional Review Board (IRB) guidelines. 35 mL aliquots of whole blood were layered on top of 15 mL Ficoll solution (Sigma-Aldrich St. Louis, MO) and centrifuged at 400g for 60 min at 4 °C. PBMCs were collected from the top layer and cryopreserved in 90% Gibco fetal bovine serum (FBS) and 5% dimethyl sulfoxide. Batch verification was performed for each donor to ensure viability, stimulation capability and proliferation. For lymphocyte experiments, PBMCs were thawed and seeded in 24 well plates at 4 million cells/mL of complete RPMI 1640 medium (Fisher Scientific, Waltham, MA), 10% Gibco FBS (ThermoFisher Waltham, MA) and 1% penicillin–streptomycin. For monocyte experiments, PBMCs were thawed and seeded in 24 well plates (Corning, Corning NY) at 3 million cells/mL of complete RPMI 1640 medium, 6% human plasma and 1% penicillin–streptomycin. Monocytes adhered overnight and the following day, the wells were rinsed 3 times with phosphate buffered saline (PBS) to remove non-adherent cells.
Whole bone-marrow was processed according to manufacturer’s protocol to isolate mesenchymal stromal cells (Lonza, Allendale, NJ). Bone-marrow derived mesenchymal stromal cells (MSCs) were cultured in complete media composed of alpha-MEM (ThermoFisher Waltham, MA), 10% Hyclone FBS (GE Life Sciences Pittsburgh, PA), 1% penicillin–streptomycin (ThermoFisher, Waltham, MA), and 2.5 μg/L human basic fibroblast growth factor (Waisman Biomanufacturing, Madison WI). Before experiments, cells were allowed to adhere overnight. Media was changed every other day. For coculture experiments, MSCs were seeded at 50,000 cells per transwell insert for a 24-well plate (Corning, Corning NY) and allowed to adhere overnight in 0.7 mL complete medium.
Characterization of extracellular enzymes
FITC-conjugated CD39 and APC-conjugated CD73 antibodies were used to characterize extracellular expression of purinergic enzymes (BD Biosciences, San Jose, CA). Adherent cells were detached with Trypsin (ThermoFisher, Waltham, MA) and PBMCs were centrifuged for 5 min at 500g. The monoclonal antibodies were incubated in 1 mL cell suspension for 10 min protected from light and rinsed 2 times with 2% bovine serum albumin in PBS. Flow cytometry was performed using BD FACS Canto II and FACS Diva software (BD Biosciences, San Jose, CA). Data was analyzed using FlowJo software (Tree Star, Ashland, OR). Positive staining was gated based on negative control cells that do not express CD39 or CD73.
ATP hydrolysis experiments
To expose cells to extracellular ATP, adenosine 5′-triphosphate disodium salt hydrate (Sigma-Aldrich, St. Louis, MO) was reconstituted in sterile, distilled water at a concentration of 50 mM, aliquoted and stored at – 20 °C. Adherent cells were seeded in various well plates at a density of 25,000 cells/cm2 and allowed to adhere overnight. Well plate sizes used were 6, 12, 24, 48 and 96-well. The following day, exposure media was prepared by diluting the ATP solution in media at concentrations ranging from 16 to 560 µM (nmol/mL). Culture media was aspirated and replaced with exposure media.
Time 0 began when soluble ATP containing-media was added to the cells. Then, after each hour of exposure, 10 µL from each well was sampled and transferred to a 96-well plate. A luciferase reagent was optimized to contain 10 mM d-Luciferin substrate (Fisher Scientific, Waltham, MA), 14 mg/mL Renilla Luciferase (Sigma-Aldrich, St. Louis, MO), 100 mM magnesium chloride (Sigma-Aldrich, St. Louis, MO), and sterile, distilled water. 95 µL of the luciferase reagent was added to each well of the 96-well plate, protected from light and shaken for 10 s, before being read using the VarioSkan plate reader (ThermoFisher, Waltham, MA) with luminescence parameters and ATP standards and controls. Because of the short half-life of ATP, standards were used at each timepoints and used to convert relative light units to nmol/mL of ATP. The amount hydrolyzed in each well was calculated by subtracting the remaining amount in individual wells at each timepoint from the initial concentration.
To determine the amount of adenosine being produced as a product of ATP hydrolysis, a fluorometric adenosine assay kit was used according to manufacturer’s protocol (Abcam, Cambridge, UK). In brief, 50,000 MSCs were seeded in a 24-well plate and allowed to adhere overnight. Cells were dosed with 500 nmol of ATP and supernatant was sampled starting after 30 min to 2.5 h of exposure and assayed in duplicate for adenosine concentration.
To correlate ATP hydrolysis with CD39 and CD73 enzymatic activity, ENTPD-1&2 inhibitor POM-1 (Tocris Bio, Bristol, UK) was used at a concentration of 100 µM. Cells were treated for 20 min with POM-1 solution and was removed before ATP dosing. Inhibition of hydrolysis was measured using the adenosine assay rather than ATP assay, as POM-1 is known to interfere with luciferase-based ATP assays [24].
Non-linear regression models
Using the ATP hydrolysis over time, the total amount hydrolyzed after 90 min was used to determine the rate of hydrolysis for different substrate concentrations. Using non-linear regression fit in Matlab, data was fit to a Michaelis Menton model for each of the population sizes to determine the maximum hydrolysis rate (Vmax) and substrate concentration at half the maximum (Km) when cells were seeded in 6, 12, 24, 48 and 96-well plates. The Vmax values were plotted as a function of the normalized cell population size (*mL/cm2) and fit with a linear curve.
Lymphocyte experiments
PBMCs were thawed and stained with 2.5 µM CFSE (ThermoFisher, Waltham, MA), a cell permeable fluorescent staining dye, which dilutes in intensity as cells proliferate, at 2 million cells/mL for 5 min protected from light. After 2 PBS rinses, cells were seeded in 24-well plates with a total volume of 0.6 mL complete media and cultured overnight in 24-well plates at 2 million cells/mL. The following day, they were stimulated with 500 ng/mL and 250 ng/mL of anti-CD3 and anti-CD28 monoclonal antibodies respectively (Fisher Scientific, Waltham, MA), and a range of soluble ATP from 0.5 to 6 mM.
For co-culture wells, 50,000 MSCs were allowed to adhere overnight in transwell inserts and added to the PBMC cultures at the time of stimulation with anti-CD3/CD28 and ATP. After 8 h, the inserts were removed, and anti-CD3/CD28 stimulation of the PBMCs continued.
96 h after stimulation cells were centrifuged and dead cells were stained with ethidium homodimer-1 (Fisher Scientific, Waltham, MA). A control well for dead cells was used by exposing PBMCs to 50% ethanol solution for 10 min before staining. After 2 PBS rinses, cells were resuspended in 2% bovine serum albumin in PBS and stored on ice. All flow cytometry was performed using the BD Biosciences Canto II. An unstimulated control was used to gate non-proliferative cells and proliferation was determined by a shift from the control as a percentage of cells. A negative control of HEK293 cells were seeded in transwell inserts at the same density of MSCs to show that effects were due to purinergic enzyme expression. Analysis was done in FlowJo and lymphocytes were gated using forward and side scatter plots. Dead cells were gated based on the cytotoxic control.
Monocyte experiments
Monocytes adhered overnight and the following day, cells were stimulated with 1× lipopolysaccharide solution (LPS) (ThermoFisher, Waltham, MA) for 4 h. Soluble ATP ranging from 0.5 to 3 mM was then added to wells for an additional 2 h. For co-culture wells, MSCs in transwell inserts were added to the monocyte cultures at the time of LPS stimulation at a density of 50,000 cells per transwell. After 2 h of ATP stimulation, inserts were removed, and the supernatant was collected and stored at – 20 °C until analysis was performed.
To measure the IL-1beta secretion, a sandwich ELISA was performed according to manufacturer protocols (R&D Systems, Minneapolis, MN). 100 μL of sample was added at 1:1, 1:10 and 1:50 dilutions in duplicates and a standard curve fit to a 4-parameter logistic curve was used to determine the concentration of IL-1β in each well.
Statistics
All experiments were done in at least triplicate (n = 3) and assay duplicates were used in plate reader experiments such as luminescence and ELISA. For kinetic modeling, least squares regression was used to determine the correlation of fit. For the proliferation experiment, statistical significance was determined using a one-way ANOVA with Tukey’s post hoc analysis. Significance was denoted for p < 0.10 and p < 0.05. To determine statistical significance between monocyte IL-1β secretion, a one-tailed, unpaired t-test was used with significance p < 0.10. All errors bars are represented as ± SD from the mean.