Intracellular cytokine staining and flow cytometry

ICS was performed as previously described [20, 49]. 5 x 10^5 PBMC were stimulated with the corresponding SARS-CoV-2 peptide at a final concentration of 10 μg/ml before blocking the secretion with 5 μg/ml Brefeldin A (Sigma Aldrich) one hour after stimulation. Cells were then incubated at 37°C overnight and stained with the Zombie NIR Fixable Viability kit for live cells as well as surface antibodies against anti-CD3 (clone: Okt3; AlexaFluor 700), anti-CD4 (clone: SK3; PerCP-Cy5.5) and anti-CD8 (clone: RPA-T8; Brilliant Violet 786) (all antibodies by BioLegend). After fixation and permeabilization (eBioscience, Foxp3/Transcription Factor Staining Buffer Set), cells were stained with anti-IFN-γ-antibodies (clone: 4S.B3; PE-Texas red; BioLegend). Cells were then analyzed on a BD LSRFortessa (BD Biosciences). We defined a T cell response as positive when the percentage of CD4+ T cells within the gate for IFN-γ was three times higher than the negative control, above 0.02%, and if the population could be clearly separated from the negative control [20, 49, 51]. R10 and DMSO were added to the negative control. Fig 1A shows an exemplary ICS result of an M-specific CD4+ T cell response.

For ex vivo ICS, PBMC from COVID-19 patients were stimulated overnight with a peptide pool of the ten most frequently detected N, M and E-specific peptides (Mem_P30, Mem_P36, Ncl_P17, Ncl_P18, Ncl_P70, Ncl_P26, Ncl_P11, Ncl_P71, Ncl_P44, and Env_P12) at a final concentration of 10 μg/ml before blocking the secretion with 5 μg/ml Brefeldin A (Sigma Aldrich) one hour after stimulation. Cells were then stained for IFN-y (clone: 4S.B3; PE-Texas red; BioLegend), TNFα (clone: Mab11; BV605; BioLegend) and IL-2 (clone: MQ1-17H12; BUV737; BD Biosciences) as described above. The threshold for positivity for the cytokines IFN-y, TNF-a and IL-2 was set at 0.02% of all CD4+ T cells. The antibody panel used for this assay is shown in S9 Table.