Recombinant human il 15

In order to induce activation of γδT cells, PBMCs were incubated with 10 µg/ml PAM (Sigma-Aldrich, St Louis, USA) in 10% FBS RPMI-1640 plus 25 ng/ml recombinant human IL-2 (Peprotech, Rehovot, Israel) and/or 10 ng/ml recombinant human IL-15 (Peprotech, Rehovot, Israel) at 37 °C humidified cell incubator with 5% CO₂. Half of the culture medium was replaced by a fresh medium and recombinant cytokines were added every 3 days. The purity of γδT cells was examined on days 0, 4, 7, 9, and 14 of culture by flow cytometry analysis. Then cell counting was determined by Precision Count Beads™ (BioLegend, San Diego, CA, USA). Only those cells under a 14-day culture presented with a ratio >90% γδ-TCR and CD3-positive cells were considered as a successful expansion and pure enough for further experiments.
For some cultures, γδT cells were pre-treated with AT-7519 (Selleck Chemicals, Houston, TX, USA) for 24 h before further culture in a regular culture medium devoid of cytokines. Cells were then harvested for corresponding assays.

Isolation of peripheral blood mononuclear cells from healthy human volunteers was done under institutional ethics approval to the REACH Team (Dalhousie University). Blood was collected in endotoxin-free sodium heparin tubes and PBMCs were isolated using Lymphoprep (Stem Cell Technologies). PBMCs were washed twice prior resuspension in complete RPMI-1640 (supplemented with 100 µg/mL streptomycin, 100 U/mL penicillin, 10% autologous human plasma), containing recombinant human IL-2 (10 ng/mL), and recombinant human IL-15 (5 ng/mL) (Peprotech). Blood iNKT cells were expanded in 6-well plates for one week using 200 ng/mL α-GalCer and TCRβ⁺Vα24Jα18⁺ iNKT cells were sorted.

Multiscreen-IP plates (Millipore-Sigma) were coated overnight at 4°C with 2μg/mL anti-IFN-γ (clone 1-D1K, Mabtech) washed with sterile PBS, and blocked with complete RPMI-10% FBS. PBMC isolated from Neuro-PASC, COVID convalescent, and healthy control subjects were used either freshly isolated or after thawing and resting overnight in media containing 10ng/μL recombinant human IL-15 (Peprotech) at 37°C, 5% CO₂. Cells were then plated at a concentration of 2.5x10⁵ cells/well in 200μL of media and stimulated with the indicated antigen mixtures from SARS-CoV-2 at a concentration of 2μg/mL in complete RPMI medium containing 5% human AB serum (Sigma-Aldrich) and 5ng/mL IL-15. Plates were incubated at 37°C, 5% CO₂ for 20h and washed 5x with dH₂O and PBS-0.05% Tween-20 (PBS-T). 2μg/mL biotinylated IFN-γ (clone 7-B6-1, Mabtech) diluted in PBS-10% FBS (PBS-F) was added to the respective wells and plates were incubated for 1.5h at RT. Plates were subsequently incubated for 40 minutes at RT in streptavidin-alkaline phosphatase in PBS-F (Jackson ImmunoResearch) was added after washing plates 5x in PBS-T. ELISPOT plates were developed using an Alkaline Phosphatase Conjugate Substrate Kit according to manufacturer’s instructions (Bio-Rad Laboratories, Carlsbad, CA). IFN-γ producing cells were quantified using an ImmunoSpot plate reader (Cellular Technologies, Ltd., Shaker Heights, OH).

The Seahorse XFe96 Analyzer (Agilent Technologies, Santa Clara, CA, USA) was used to measure the glycolytic function of NK-cell subsets. Educated and uneducated NK cells were FACS-sorted on a BD FACSAria II SORP flow cytometer using enriched NK cells as primary material. Sorted NK cells were cultivated overnight in complete cell culture medium (RPMI 1640 Medium, Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% (v/v) heat-inactivated FBS (Biochrome, Berlin, Germany) and 5 ng/ml recombinant human IL-15 (PeproTech, Hamburg, Germany). The following day 2 × 10⁵ to 1 × 10⁶ NK cells were resuspended in glucose-free assay medium prior to the analysis and then into a 96-well Seahorse XF cell culture microplate (Agilent Technologies, Santa Clara, CA, USA) and incubated for 30 min in a CO₂-free incubator at 37°C. Sample replicates were used whenever sufficient cell numbers were available (max. triplicates). Subsequently, oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were measured in a Glycolysis Stress Test (Agilent Technologies, Santa Clara, CA, USA) using the manufacturer's protocol.