Perforin apc

Peripheral blood mononuclear cells (PBMCs) were isolated from the fresh blood of patients using Ficoll density gradients as described previously [16 (link)]. Isolated PBMCs were stained for surface markers, fixed, permeabilised with IntraPreReagent (Beckman Coulter, Fullerton, CA) and further stained with antibodies directed against intracellular markers. Leukocytes were stimulated with Leukocyte Activation Cocktail (BD Bioscience, USA) at 37 °C for 4 h prior to intracellular staining using the manufacturer’s staining protocol. Anti-human mAbs against CD3-PE-CF594, CD56-FITC, NKG2D-PE, NKp46-PE-CY7, NKp-30-APC, NKp44-PE, NKG2A-APC, CD69-PE-CY7, PD1-Pacific blue, Tim-3-APC, perforin-APC, Granzyme B-BV421, IFN-γ-PE, and TNF-α-PE with corresponding isotype-matched controls were purchased from BD Biosciences (San Jose, CA, USA). Data were acquired on a Gallios instrument (Beckman Coulter, Brea, CA, USA) and analysed using FlowJo software (Flow jo, LCC, USA).

For flow cytometry, the stimulated PBMCs were stained for viability and surface markers (CD4 FITC, 1:100; CD8 BV510, 1:100; CD69 PE-CF594 1:100; CD137 BV-605 1:100; OX-40 PE-Cy7 1:100; all BD Biosciences) in FACS buffer (PBS supplemented with 2% FBS (Gibco, Invitrogen^TM, USA)) for 40 min at 4 °C. Next, cells were fixed and permeabilized using the Cytofix/Cytoperm kit (BD Biosciences, San Diego, CA, USA), according to the manufacturer’s instructions. Intracellular staining was performed by incubating the cells in perm/wash buffer supplemented with antibodies for 30 min at 4 °C (IFNγ PE, 1:50; IL-2 BV711, 1:50; TNFα BB700, 1:50; GranzymeB (GZB) PE 1:50; Perforin APC 1:100; all BD Biosciences, San Jose, CA, USA). Samples were acquired on a fluorescence-activated cell sorter (FACS) Symphony^TM instrument (BD Biosciences), using BD FACSuite software version 1.0.6 (BD Biosciences, San Jose, CA, USA), and analyzed with FlowJo software version VX (Tree Star, San Carlos, CA, USA). Functional profiles were deconvoluted by employing Boolean gating in FlowJo version XV (Tree Star, San Carlos, CA, USA) (Figure S1).

Cells were prepared in a single-cell suspension and washed with phosphate-buffered saline. After incubation with FcR Blocking Reagent (miltenyi, 130-059-901) for 10 min, NK cells were stained with fluorescence antibodies for 30 min at 4°C. The following antibodies were used in this research (all antibodies from BioLegend unless otherwise indicated): FITC-conjugated antibody to human CD3 (300306), CD56-PE (304606), CD107a-APC (328620), PD-1-APC (BD Pharmingen, 558694), Perforin-APC (353311), IFN-γ-APC (502512), CFSE, and PI (BD Pharmingen). GCMSCs were assessed for membrane expression of selected markers by using CD19-FITC, CD29-PE, CD34-FITC, CD45-FITC, CD90-PE, and CD105-PE antibodies (all antibodies above from eBioscience). For intracellular cytokine staining, NK cells were stimulated for 5 h with a 2 μl/ml cell stimulation cocktail (plus protein transport inhibitors) (eBioscience) containing phorbol 12-myristate 13-acetate, ionomycin, brefeldin A, and monensin. Then, cells were stained for surface markers, fixed, and permeabilised with eBioscience FoxP3 fixation buffer according to the manufacturer's instructions. The fixed cells were then stained with perforin, CD107a, and IFN-γ.