Immunospot

IFN-γ pre-coated ELISpot plates (Mabtech) were blocked with complete medium (RPMI and 5% human AB serum) before 300,000 PBMCs were seeded per well and stimulated for 18 h with 2 µl per well of PepTivator SARS-CoV-2 S, M or N pools (representing 1 µg ml⁻¹ final concentration per peptide; Miltenyi Biotec). Plates were developed with human biotinylated IFN-γ detection antibody (7-B6-1-ALP, 1:200), followed by incubation with BCIP/NBT Phosphatase Substrate (SeraCare). Spot-forming units (SFU) were quantified with ImmunoSpot (Mabtech). To quantify positive peptide-specific responses, spots of the unstimulated wells were subtracted from the peptide-stimulated wells, and the results were expressed as SFU/106 PBMCs.

Elispot analysis was performed after 3 days of DC/T-cell co-culture. After that CD4⁺/OT-II T-cells were counted and plated again over night at 37°C in a 96-well/ELISpot-plate (Cellular Technology Limited, CTL, ImmunoSpot, Cleveland, USA), with 6 h-LPS-treated DC-OVA, just OVA or TRP as controls, and PMA+Ionomycin. To monitor unspecific signals and background, T-cells, DC-OVA, DC-TRP, Lcn2^−/− DCs and WT DCs, were also co-cultured. We used ELISpot plates (Millipore, Massachusetts, USA) for IFN-γ, IL-10 and IL-17A (from Mabtech, Sweden), following the ImmunoSpot protocol and recommendations. Spots were counted using ImmunoSpot 5.0 software. Histograms are the results of the spots from T-cell+WT DC-OVA or T-cell+Lcn2^−/− DC-OVA calculated after subtracting the spots of the controls.