Preparation of PBMCs and stimulation with SARS-CoV-2 overlapping peptide pools

Peripheral blood mononuclear cells (PBMCs) were prepared from whole blood by gradient centrifugation as previously described [27]. Isolated PBMCs were stimulated with 15mer overlapping peptide pools (OPPs) from SARS-CoV-2 proteins with an overlap of 11 amino acids. SARS-CoV-2 PepTivator peptide pools (Miltenyi Biotec, Germany) were used containing overlapping peptides spanning in silico predicted immune dominant parts of the S-protein [28], or, covering the complete sequence of the N- and M-proteins. Peptide pools were dissolved per the manufacturer’s instructions in sterile water and used at a concentration of 1 µg/ml. 2.5 × 10⁶ PBMCs were plated for each condition in 96-U Well Plates in RPMI media (Life Technologies, USA), supplemented with 1% Penicillin–Streptomycin–Glutamine (Sigma Aldrich, USA) and 10% FCS (PAN-Biotech, Germany) and were stimulated or left untreated as a control for 16 h. As a positive control, cells were stimulated with SEB (1 µg/ml, Sigma Aldrich) and negative control was with vehicle (a medium to dissolve peptide pools). After 2 h Brefeldin A (5 µg/ml, Sigma Aldrich, USA) was added. T cells stimulated with SARS-CoV-2 OPPs were stained with optimal concentrations of antibodies for 10 min at room temperature in the dark. Stained cells were washed twice with PBS/BSA before preparation for intracellular staining using the Intracellular Fixation & Permeabilization Buffer Set (Thermo Fisher Scientific, USA) as per the manufacturer’s instructions. Fixed and permeable cells were stained for 30 min at room temperature in the dark with optimal dilution of antibodies against intracellular antigens. All samples were immediately acquired on a CytoFlex flow cytometer (Beckman Coulter, USA). Antigen-specific responses were considered positive after non-specific background was subtracted and more than 0.01% cells were positive. Negative values were set to zero. Quality control was performed daily using the recommended CytoFLEX Daily QC Fluorospheres (Beckman Coulter, USA). No modification to the compensation matrices was required throughout the study. As a result of low cell counts after thawing, only PBMCs from 11/20 renal transplantation patients and 19/20 healthy controls could be stimulated with the M protein.