RPMI-1640 Dutch modified (Gibco, Massachusetts, United States) culture medium, containing sodium bicarbonate and 20 mM HEPES, supplemented with penicillin/streptomycin (100 U/ml), sodium pyruvate (1 mM), glutamine/glutamax, and 10% human pooled serum (HPS, Radboudumc), was used in all experiments. After cell isolation, 2.5 × 104 cells/well were cultured in 96-well U-bottom plates and stimulated with Dynabeads® Human T-Activator CD3/CD28 (αCD3/CD28 beads, 1:5 of bead:cell ratio) (Gibco, Massachusetts, United States) in the presence of recombinant human (rh) IL-2 (rhIL-2, 100 U/ml) (Proleukin Prometheus Laboratories, California, United States). In some conditions, cultures were supplemented with rhTNFα (50 ng/ml, R&D, Minnesota, United States), or TNFα inhibitors etanercept (5 μg/ml; ETN—Enbrel, Pfizer, New York, United States), or TNFR2 agonist (2.5 μg/ml, Clone MR2-1, Hycult Biotech, Uden, the Netherlands). To examine the effect of a pharmaceutical inhibitor, tofacitinib (0.112 μM, Pfizer, New York, United States), PKC inhibitor Sotrastaurin (1 μM), Lck inhibitor A420983 (1 μM), or p38α/β kinase inhibitor UR13870 (10 μM) was pre-incubated with the FACS-sorted cells for 30 min before the addition of any stimulus. In some cases, cells were stimulated with PMA (12.5 ng/ml) and ionomycin (500 ng/ml) for 20 h.
Tofacitinib
Manufactured by Pfizer
Sourced in United States
Tofacitinib is a pharmaceutical product manufactured by Pfizer. It functions as a Janus kinase (JAK) inhibitor, which is a class of medications that work by blocking the activity of one or more of the Janus kinase enzymes.
Automatically generated - may contain errors
Lab products found in correlation
Il 22, by R&D Systems (2 mentions)
Iscove s modified dulbecco s medium, by Thermo Fisher Scientific (2 mentions)
Celltrace violet, by Thermo Fisher Scientific (2 mentions)
Glutamax, by Thermo Fisher Scientific (2 mentions)
Penicillin, by Thermo Fisher Scientific (2 mentions)
Streptomycin, by Thermo Fisher Scientific (2 mentions)
Etn enbrel, by Pfizer (1 mentions)
Penicillin streptomycin, by Merck Group (1 mentions)
Rpmi 1640 medium, by Merck Group (1 mentions)
Interleukin 2 (il 2), by Novartis (1 mentions)
Venetoclax, by Selleck Chemicals (1 mentions)
Rag2 mice, by Jackson ImmunoResearch (1 mentions)
Pf 06651600, by Pfizer (1 mentions)
Balb c, by Jackson ImmunoResearch (1 mentions)
Matching placebo, by Pfizer (1 mentions)
Phytohemagglutinin (pha), by Thermo Fisher Scientific (1 mentions)
Phytohemagglutinin (pha), by Merck Group (1 mentions)
Recombinant human il 2, by R&D Systems (1 mentions)
Il 15, by R&D Systems (1 mentions)
Ficoll hypaque gradient, by Axis-Shield (1 mentions)
15 protocols using tofacitinib
1
T-cell Activation and Modulation Assay
2
Characterization of Malignant T Cell Lines
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The malignant T cell lines, MyLa2059 and SeAx, were derived from MF skin lesions and PBMC's from SS patients, respectively, and have been previously characterized [74 (link)]. Both cell lines were cultured in RPMI1640 medium (Sigma-Aldrich) supplemented with 5% penicillin/streptomycin (Sigma-Aldrich). In addition, the medium was supplemented with 10% Fetal Bovine Serum for MyLa2059 cells and 10% Human Serum for SeAx cells. The SeAx cells were cultured in the presence or absence of IL-2 (103U/mL IL-2, Novartis). Peripheral blood mononuclear cells (PBMCs) were isolated from a patient diagnosed with SS using a Ficoll Gradient, Lymphoprep (Axis-Shield PoC AS), as previously described [17 (link)] and cultured as described for SeAx cells. JAK3 inhibitor, Tofacitinib (CP-690550, Pfizer), was used in experiments with MyLa2059 and SeAx cells.
3
JAKinibs and Venetoclax Administration in Mice
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BALB/c and Rag2−/− mice were purchased from Jackson Laboratory. All animal studies were performed according to NIH guidelines for the use and care of live animals and were approved by the NIAMS Institutional Animal Care and Use Committee. JAKinibs were resuspended in 0.5% methyl cellulose and animals were dosed orally twice daily with vehicle or 30 mg/kg of tofacitinib (kindly provided by Pfizer) or 20 mg/kg of PF-06651600 (provided by the National Center for Advancing Translational Sciences (NCATS), NIH) for 1 week (or 3 days, where indicated) (27 (link)). ABT-199 (Venetoclax, Selleckchem) was resuspended in 60% Phosal 50PG, 30% PEG 400, and 10% EtOH. Animals were dosed orally once a day with vehicle or 90 mg/kg for a week.
4
Tofacitinib vs Placebo for Disease
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Eligible participants were randomized in a 2:1 ratio to either 5 mg twice a day of tofacitinib or matching placebo (provided by Pfizer Inc.). A randomization schedule using computer-generated block randomization with the random block sizes (known only by the statistician) was used to randomize patients. The study staff, including the research pharmacists, and participants were blinded to the treatment assigned.
5
PBMCs Isolation and T Cell Activation
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Venous blood was collected in EDTA tubes and PBMCs were isolated using a Ficoll-Hypaque gradient according to standard protocol (Axis-Shield Diagnostics, Dundee, UK). PBMCs were labeled with CellTrace Violet (ThermoFisher Scientific, Bleiswijk, the Netherlands) and were stimulated with phytohemagglutinin (PHA, 5 µg/ml, ThermoFisher Scientific), ConA (10 µg/ml), anti-CD3 (0.5 µg/ml, Sanquin, Amsterdam, the Netherlands) or anti-CD3/CD28 stimulator beads (0.5 bead per PBMC) with or without recombinant human IL-2 (1, 10 or 100 IU/ml, R&D Systems, Minneapolis, MN, USA) or IL-15 (1, 10 or 100 µg/ml, R&D Systems) for the indicated time-points. In some experiments, tofacitinib (Pfizer, New York, NY, USA; 200 or 1000 µM) was added to the anti-CD3/CD28 stimulated conditions. Cells were cultured in Iscove’s modified Dulbecco’s medium (ThermoFisher Scientific) supplemented with heat-inactivated fetal calf serum, Glutamax (ThermoFisher Scientific), 2-mercaptoethanol, penicillin, and streptomycin. For retroviral viral transduction, PBMCs from a healthy donor were isolated and T cell blasts were generated by culturing PBMCs with 1 µg/ml PHA (Sigma-Aldrich, Zwijndrecht, The Netherlands) for 72 h in RPMI-1640 medium supplemented with 10% human serum (#H4522, Sigma-Aldrich) and expanded in culture with 100 IU/ml recombinant human IL-2 (R&D Systems).
6
Evaluation of JAK Inhibitors
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Baricitinib was kindly provided by Eli Lilly (Indianapolis, IN, USA). Tofacitinib was kindly provided by Pfizer (New York, NY, USA). Anti-interleukin (IL)-6 receptor α antibody, tocilizumab, was purchased from Chugai Pharmaceutical Co. (Tokyo, Japan).
7
Modulating Autophagy and Apoptosis in Fibroblast-Like Synoviocytes
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For in vitro experiments, FLS were grow in a Dulbecco’s modification of Eagle medium (DMEM) supplemented with 10% Fetal Bovine serum (FBS, EuroClone) heat inactivated at 50°C for 30′, 50 IU/ml penicillin/streptomycin, 2 mM glutamine, and 10 mM HEPES, in a controlled atmosphere incubator at 37°C. Cells were subjected to different treatments.Supplementary Figure S1 ).
The study was approved by the Ethics Committee of Sapienza University of Rome (protocol number 707/17).
- tofacitinib (provided by Pfizer) resuspended in DMSO and then diluted in cell medium at a concentration of 1 μM;
- rapamycin resuspended in DMSO and then diluted in cell medium at a concentration of 200 nM (Sigma-Aldrich);
- nutrients deprivation (starvation) obtained by culturing cells at a lower serum concentration (2% FBS).
The study was approved by the Ethics Committee of Sapienza University of Rome (protocol number 707/17).
8
Modulation of Entheseal Cell IFNα
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After digestion, 1×105 cells/ml entheseal cells were seeded into 96-well plate. Cells were stimulated with ODN as before, with and without tofacitinib 1 μM (Pfizer), PDE4i/Rolipram 100µM (Cayman Chemical) or Methotrexate 5 mg/ml (Cayman Chemical) for 24 hr. DMSO 0.1% served as a solvent control. IFNα was quantified by ELISA as before.
9
Cucurbitacin and JAK Inhibitor Preparation
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Cucurbitacin E (NSC 106399, Sigma-Aldrich, Saint Louis, MO), cucurbitacin I (NSC 521777, Indofine Chemical Company, Hillsborough, NJ) and Stattic (Calbiochem, San Diego, CA) were dissolved in DMSO to a concentration of 15 mM, 50 mM and 100 mM respectively. JAK inhibitors (JAKi) AZD1480 (Tocris, Bristol, UK), decernotinib (MedChem Express, Monmouth Junction, NJ), ruxolitinib (Selleck, Houston, TX) and tofacitinib (Pfizer, Capelle a/d Ijssel, The Netherlands) were dissolved in DMSO to a concentration of 10 mM. All compounds were stored at −20 °C. During the experiments, compounds were diluted in culture medium to the final concentrations.
10
JAK Inhibitor Effects on B Cells
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Peripheral blood mononuclear cells (PBMCs) were isolated from buffy coats or EDTA blood by density gradient centrifugation. Total or CD27+ B cells were isolated magnetically using EasySep isolation kits (Human B-cell Isolation Kit/CD27+ B-cell Isolation Kit (Stemcell Technologies)) following the manufacturers’ instructions. B cells were plated in 96-well plates with 30.000 cells/well in Iscove’s Modified Dulbecco’s Medium (Life Technologies) supplemented with 10% FCS, insulin, apo-transferrin, non-essential amino acids, glutamine and glutathione as described previously (20 (link)) and stimulated with CpG (ODN2009, Apara Biosciences) at a concentration of 0.5µM. Baricitinib (Toronto Research Chemicals), tofacitinib (provided by Pfizer Inc, Peaback, USA), ruxolitinib (Novartis), upadacitinib (ABT-494, MedChemExpress), filgotinib (GLPG0634, selleckchem) were stored as 10nM stocks in dimethylsulfoxide and further diluted in medium as needed. JAK inhibitors were added to indicated samples at doses of 10-3000nM.
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