Cd8 af700

Cells isolated from spleen, lungs or peripheral blood were cultured with 2 µg/ml of two immunodominant peptides (Pepscan, Lelystad, The Netherlands), [SSTHEANTMAMMARDT] and [AGYAGTLQSLGAEIAV] of the TB10.4 protein, previously demonstrated to stimulate both CD4⁺ & CD8⁺ T cell responses (Kaveh & Hogarth, unpublished); 1 µg/ml anti-CD28 (BD Biosciences) and 10 µg/ml Brefeldin A (Sigma) for 16 h at 37 °C/5% CO₂. Cells were washed at 300 g for 5 min and surface stained with combinations of CD62L-FITC, CD27-PerCP-Cy5.5, CD8-AF700, CD44-BV421, CD127-PE-Cy7, CD69-FITC, CCR7-BV421, live/dead-Zombie Aqua (all BioLegend) and CD4-APC-H7 (BD Biosciences). Cells were then washed, treated with BD Biosciences Cytofix/Cytoperm as per manufacturer’s instructions and stained intracellularly with combinations of IFN-γ-PE-Cy7, IL-2-APC (both eBioscience), IFN-γ-BV605 and TNF-α-BV605 (both BioLegend). Cells were washed again and analysed using an LSRFortessa™ analyser utilising a 532 nm laser for PE and PE-conjugate excitation with FACSDiva™ software (BD Biosciences). Final analysis was performed using FlowJo® software (Tree Star Inc.) on a minimum of 100,000 live lymphocytes (50,000 for peripheral blood).

Supernatants freshly derived from killing assay were immediately adopted for chemotaxis assays. 600 μl of supernatant were placed in the bottom chamber of Corning^® Transwell^® polycarbonate membrane cell culture inserts 6.5 mm Transwell with 5.0 μm pore polycarbonate membrane insert (CLS3421). CD3-CD28-activated CD8⁺ T cells were labeled with 2.5 μM of CellTracker^™ Green CMFDA Dye, then resuspended at 1*10⁶ cells/ml in CST^™OpTimizer T cell expansion SFM medium (Thermo Fisher, A1048501) without serum. 100 μl of cell suspension was then seeded in the upper chamber of the transwell. After 3 hours of migration, cells migrated to the bottom chamber were collected and stained with CD8 AF700 (BioLegend) and resuspended in 200 μl of PBS. 150 μl of cell suspension was then acquired at constant flow rate (2.5 μl/sec) on a BD 5 laser LSR Fortessa. Data were analyzed using FlowJo V.10. % of migrated cells has been calculated by applying this formula: (Number of cells in the bottom chamber/Number of cells seeded in the upper chamber) x100.
We then calculated the fold change of migration as compared to the untreated conditions (spontaneous migration).

PBMCs (1 × 10⁶) were stained with fixable blue dead cell stain (ThermoFisher, Waltham, Massachusetts and USA) or Zombie NIR™ Fixable Viability Kit (Biolegend, London, UK). This was followed by washes and surface marker staining with antibodies CD4-BUV395, CD8-AF700, CD19-AF488, and CD14-BV711 (Biolegend, London, UK) followed by subsequent washes. Following surface staining, to determine the expression of membrane lipid rafts cells were subsequently stained with a surrogate lipid raft marker CTB¹⁶, conjugated to FITC (1:100 dilution in PBS) (Sigma, Dorset, UK) followed by subsequent washes and fixation in 2% PFA before running on the flow cytometer. Altneratively, cells were fixed filipin complex (Sigma) as described previously [74 (link)]. Stains and washes were carried out in cell staining buffer (Biolegend). Appropriate unstained controls were used for lipid stains.
Data was acquired using a LSRFORTESSA X-20 (BD, Wiltshire, UK) flow cytometer and analysis performed using FlowJo Single Cell Analysis Software (TreeStar). Application settings were created and Cytometer Setup and Tracking (CS&T) (BD) beads were run to assess cytometer performance for each experiment.

PBMCs were resuspended in AIM-V media (no FBS/AB serum) and seeded in 24-well ultra-low attachment plates (Corning) at a density of 1 × 10⁶ cells/mL and incubated for 1 hour at 37°C to recover. Next, cells were left unstimulated in AIM-V medium or stimulated with 1000 U/mL of recombinant human IFN-β for 16 hours. Finally, Brefeldin A (1 ug/mL, Sigma) and Monensin (2 µM, Biolegend) were added to the culture media and incubated for an additional 2 hours.
Following incubation, cells were labeled with Zombie Aqua Viability Dye (BioLegend) for 20 minutes at 4°C in the dark, washed with FACS buffer, and stained with a surface antibody cocktail containing CD3-PE/Cy7 (BioLegend, clone UCTH1, 1:400), CD4-APC (BioLegend, clone RPA-T4, 1:100), CD8-AF700 (BioLegend, clone RPA-T8), and CD11c-PE/Dazzle594 (BioLegend, clone Bu15), for 15 minutes at 4°C in the dark. For intracellular staining, the Foxp3 Staining Kit (Thermo Fisher Scientific) was used according to the manufacturer’s instructions. Cells were fixed with Foxp3 Fixation/Permeabilization buffer for 30 minutes at room temperature and then washed with 1× permeabilization buffer. Cells were stained for 45 minutes at room temperature with ISG15-PE (R&D Systems, clone 851701) in 1× permeabilization buffer. Data were acquired on the Symphony A3 flow cytometer (BD, Biosciences) and analyzed using FlowJo v.10.

Splenocytes were incubated with either 20 ng mL⁻¹ phorbol myristate acetate (Sigma‐Aldrich) and 1 ug mL⁻¹ ionomycin (Cell Signaling Technology), S protein‐derived peptides (GenScript), N protein‐derived peptides (GenScript), or control (no stimulation) in 1X Brefeldin A and 1X Monensin (Biolegend) solutions for 6 h at 37 °C. Next, the cells were washed with PBS and incubated for 30 min with Fixable Viability Dye eFluor 780 (eBioscience) in PBS (1:1000 dilution) at 4 °C. Cells were then washed once with PBS and twice with PBA before being stained overnight with fluorochrome‐conjugated antibodies (CD3‐FITC (Clone 145‐2C11), CD4‐PerCP‐Cy5.5 (Clone GK1.5), CD8‐AF700 (Clone 53–6.7), CD44‐BV605 (Clone IM7), Biolegend) in PBA at 4 °C. Cells were subsequently washed 3 times with PBA, fixed in 4% PFA, and then permeabilized using a Cyto‐Fast Fix/Perm Buffer Set (Biolegend) according to the manufacturer's protocol. Intracellular staining was performed by incubating the cells with fluorochrome‐conjugated antibodies (IL‐13‐PE (Clone: W17010B), IL‐4‐PE‐Cy7 (Clone: 11B11), IL‐17‐APC (Clone: TC11‐18H10.1), IL‐5‐BV421 (Clone: TRFK5), IFNγ‐BV510 (Clone XMG1.2), Biolegend) in permeabilization buffer for 30 min at 4 °C. Lastly, cells were washed 3 times with permeabilization buffer, resuspended in PBA, and analyzed using the Attune NxT flow cytometer (Thermo Fisher Scientific).