Human th cytokine panel

For intracellular cytokine production, 3 × 10⁵-irradiated (80 Gy) T2 cells were loaded for 2 h with 10 μM peptide and co-cultured for 18 h with 3 × 10⁵ TCR-transduced human T cells in the presence of 1 μg/ml brefeldin A (Sigma-Aldrich) in a total volume of 250 μl of culture medium per well in round-bottom 96-well plates. Cells were then surface stained for CD8 and CD19, fixed/permeabilized using a Fixation/Permeabilization Solution Kit (BD Biosciences), and stained intracellularly for IFNγ and IL-2. Data were acquired using an LSRFortessa and analyzed with FlowJo software. For extracellular cytokine secretion, 1 × 10⁵-irradiated T2 cells were loaded for 2 h with the indicated concentrations of peptide and co-cultured for 18 h with 1 × 10⁵ TCR-transduced human T cells in round-bottom 96-well plates containing 250 μl of culture medium per well. Supernatants were harvested from duplicate wells and tested for IFNγ and IL-2 using human ELISA Kits (BD Biosciences). Absorbance was read at 450 nm. A similar experimental set-up was used for the LEGENDplex assay (BioLegend), with the exception that irradiated T2 cells were loaded with 10 μM peptide, and supernatants were tested for secreted cytokines using a Human Th Cytokine Panel (BioLegend).

Cell culture supernatants and/or sera samples collected from COVID-19 patients and healthy volunteers were analyzed using the LEGENDPlex, Human Th Cytokine Panel and Human Inflammatory Panel (both 13‐plex, BioLegend) and the following cytokines were analyzed this way: IL-10, IL-9, IL-6, IL-2, IL-13, IL-5, IL-22, IL-21, IL-4, IL-17F, IL-17A, TNF-α, IFN-γ, IL-1β, IFN-α, MCP-1, IL-8, IL-12p70, IL-18, IL-23, IL-33, according to manufacturer’s instructions. Besides these cytokines, TGF-β, IL-33, and IL-1β concentrations in sera samples were determined by Duo Set ELISA test (R&D Systems, Minneapolis, MN, USA), according to manufacturer’s protocol. The levels of SARS-CoV-2 specific IgG and IgM antibodies were determined by commercial ELISA kits (BioVendor, Brno, Czech Republic). The positivity/negativity of the sera for IgM and IgG was defined according to manufacturer’s recommendations.