T bet clone ebio4b10

To perform surface staining, 1 × 10⁶ cells were placed in individual wells of a 96-well round bottom plate and incubated with the appropriate antibody cocktails for 15 min at 4°C on a slow rocker. After the staining, cells were fixed in a solution of 2% ultrapure formaldehyde (Polysciences, Inc., Warrington, PA, USA) in FACS buffer for 20 min on ice, washed twice, and analyzed the following day on the Canto II (BD Biosciences) or FACSCalibur (BD Biosciences). Intracellular staining was performed using Cytofix/Cytoperm Fixation/Permeabilization Solution Kit with BD GolgiStop (BD biosciences) according to the manufacturer’s instruction. Flow cytometry acquisition was performed on an LSRIISorp. Data were analyzed using FACS Express or FlowJo software (Tree Star, Inc., Ashland, OR, USA). Antibodies against CD45 (clone 30-F11, BD Pharmingen), CD3 (clone 145-2C11, BD Pharmingen), CD4 (clone GK1.5, BD Pharmingen), CD8 (clone 5H10, Biolegend), T-bet (clone eBio4B10, eBioscience), IL-17A (clone ebio17B7, eBioscience), IL-4 (clone B11B, Biolegend), IFNγ (clone XMG 1.2, eBioscience), IL-22 (clone A3.6M, eBioscience and clone poly5164, Biolegend), TGF-β (clone 11A5, Biolegend), IL-17F (clone ebio18F10, eBioscience), NKP46 (clone 29A 1.4, Biolegend), c-Kit (clone 2B8, Biolegend), Sca-1 (clone D7, BD Pharmingen), and CD127 (clone A7R34, eBioscience) were used.

Fluorochrome-conjugated and unconjugated murine anti-human α-smooth muscle actin (α-SMA, clone 1A4) monoclonal antibodies (mAbs) were purchased from Sigma (St. Louis, MO, USA). Fluorochrome-conjugated forms of IgG1_κ, IgG_2a, isotype controls, and mAbs directed against human CD90 (clone 5E10) were purchased from BD Bioscience and eBioscience (San Diego, CA, USA). Fluorochrome-conjugated antibodies against human and murine CD4 (clone RPA-T4 and RM4-5, respectively), Tbet (clone eBIo4B10), isotype controls as well as mAbs against human PD ligands, PD-L1 (clone MIH1, clone 29E.2A3), and PD-L2 (clone MIH18), antihuman IFN-γ (clone 45.B3) were obtained from eBioscience (San Diego, CA, USA) and BioLegend Inc., Alexa Fluor^® (AF^®) 488- and AF^®633-labeled donkey anti-mouse IgG_2a and IgG1_κ (respectively), Zenon Mouse IgG antibodies labeling kits were purchased from Life Science Technology, Inc. (CA, USA). Functional grade anti-human IL-4 mAbs (clone B-S4), recombinant human IL-12 and IL-2 were purchased from ThermoFisher Scientific.