Golgistop

For assessment of T cell intracellular cytokines, LN single-cell suspensions were prepared by gently pressing LN through a 70 µm cell strainer, washed with IMDM, and cultured in fetal calf serum-supplemented IMDM (Invitrogen) in the presence of 50 ng/mL PMA (Sigma-Aldrich), 1 µg/mL ionomycin (Merck Millipore) and 1 µL/mL GolgiStop™ (BD Pharmingen) for 5 h at 37 °C.
DC were prepared from auricular LN by digesting with 100 µg/mL Liberase TL and 100 µg/mL DNase I (both from Roche, Germany) for 25 min at 37 °C before passing through a 70 µm cell strainer. For assessment of cytokine production by DC, monocytes, and NK cells, single-cell suspensions were incubated in fetal calf serum-supplemented IMDM in the presence of GolgiStop™ (BD Pharmingen) and Brefeldin A (Sigma Aldrich) for 6 h.

PBMCs and dMCs were thawed and rested overnight in low dose IL-15 as described above. For stimulation by PMA/Ionomycin, 1 × 10⁶ mononuclear cells were treated with eBioscience™ cell stimulation cocktail (Invitrogen) for 4 h in 96-well U bottom plates at 37 °C, 5% C0₂. Golgi Plug (BD Biosciences) and Golgi Stop (BD Biosciences) were added for the final 3 h of stimulation. For K562 stimulations, mononuclear cells were co-cultured with K562 target cells at a ratio of 10:1 for 6 h in 96-well U bottom plates at 37 °C, 5% CO₂. Golgi Plug (BD Biosciences) and Golgi Stop (BD Biosciences) were added for the final 5 h of stimulation. Cells were then stained and acquired as outlined above. To analyse polyfunctional NK responses to stimulation, Lin− CD56+ subsets were manually gated on the tSNE plot to show dNK1 (c10–c13), dNK2 (c9), dNK3 (c5, c8), and pbNK (c1, c2) subsets. Boolean gating arrays were created using FlowJo to determine the frequency of cells within these subsets that stained for one, two, or three readouts. Readouts were defined as: CD107a and/or IFNγ and/or at least one of the cytokines MIP1α, MIP1β, GM-CSF, XCL1 (Cyto). Non-responding cells that expressed none of the functional readouts were excluded.

Single cell suspensions were stained for surface markers in PBS for 20 minutes at 4°C. Intracellular proteins (i.e., cytokines, FOXP3, and Ki67) were assessed using the FOXP3/Transcription staining buffer set (eBioscience, San Diego, CA) and following manufacturer’s instructions. Cells were permeabilized for 30 minutes and stained for intracellular proteins for 1 hour at 4°C. All fluorochromes were purchased from Biolegened and eBioscienes. Ex vivo re-stimulation was performed using PMA (Sigma-Aldrich), ionomycin (Sigma-Aldrich), and Golgi Stop (BD Biosciences) for 4 hours as previously described (Kornete et al., 2012 (link)). For OTI and OTII peptide re-stimulation, peptides OVA257-264 and OVA323-339, were incubated with transgenic T cells at 10μg/ml with Golgi Stop (BD Biosciences) for 6 hours followed by surface staining and ICS. Dead cells were distinguished using the fixable viability dye efluor780® from eBioscience. Single cell suspensions were fixed and permeabilized using the FOXP3 Transcription staining buffer set. Samples were acquired on the BD LSRFortessa™ and on the BD FACSymphony™. Flow cytometry data was analyzed using FlowJo (TreeStar).

Mucosal-associated invariant T cell function was determined in vitro using paraformaldehyde-fixed Escherichia coli stimulation (One Shot Top10, Life Technology, multiplicity of exposure 10) in the presence of 1.25 µg/ml anti-CD28 mAb (clone L293, BD Biosciences) (34 (link)). E. coli was fixed for 5 min in 1% paraformaldehyde. PBMCs were further cultured for 24 h at 37°C/5% CO₂ in RPMI medium supplemented with 10% FBS. Monensin (Golgi Stop, BD Biosciences) was added during the last 6 h of the stimulation. iNKT cell function was determined in vitro using α-GalCer (KRN7000, Enzo Life Science, Farmingdale, NY, USA) at 100 ng/ml. PBMCs were further cultured for 8 h at 37°C/5% CO₂ in RPMI medium supplemented with 10% FBS. Monensin (Golgi Stop, BD Biosciences) was added during the last 6 h of the stimulation.

Cells were stimulated with 50 ng/mL PMA (Cayman Chemical) and 1 µg/mL ION (Sigma-Aldrich) for 4 hours at 37 °C. After 1-hour GolgiStop (BD Biosciences) was added to all samples. For human T cell studies and CD8 re-stimulation studies, GolgiStop (BD Biosciences) was added to all samples 1 hour prior to staining. Cells were resuspended in cell staining buffer (BioLegend) and stained with FVS780 viability dye (BioLegend), Fc Block (BioLegend), and various extracellular antibodies. Cells were washed in cell staining buffer and fixed using Fixation Buffer (ThermoFisher) for 1 hour at 4 °C. Cells were then washed in Permeabilization Buffer (ThermoFisher) and stained with intracellular antibodies overnight. Cells were washed in Permeabilization Buffer and resuspended in cell staining buffer. All flow cytometry data was collected on LSR II (BD Biosciences) and analyzed using FlowJo V10 software (TreeStar). For every sample 1 × 10⁵ events were uniformly collected for all FACs analyses described.