2.7. Intracellular flow cytometry of SARS-CoV-2 specific T cells

To decipher subsets of the T cell compartment, SARS-CoV-2 specific T cells of seronegative (n=7) and seropositive (n=7) vaccine recipients were expanded and analysed by intracellular flow cytometry (ICFC).

First lithium heparin whole blood was collected and PBMCs were isolated using a Lymphoprep density gradient medium (Stemcell, Vancouver, Canada) according to the manufacturer's instructions. In brief 4 mL lithium heparin blood were diluted with 3 mL PBS, PBMCs were isolated by layering the 7mL on 4 mL Lymphoprep and subsequent density-gradient centrifugation for 30 minutes at 850 x g. PBMCs were collected from the interphase, washed twice, cryopreserved in heat- inactivated fetal calf serum (Sigma, Missouri, US) supplemented with 10% dimethylsulfoxid (Sigma, Missouri, US) and stored in liquid nitrogen until further use.

To expand SARS-CoV-2 reactive T cells, 2 × 10⁵ PBMCs in 200 µL of RPMI medium supplemented with 10% FCS were pulsed with 0•6 μg/mL spike (pepS, pepS1 and pepS+) or nucleocapsid (pepN) peptide pools in the presence of 10 U/mL interleukine-2 (IL-2). Cells were cultured with IL-2 only served as a negative control. After 60 hours cells were restimulated with or without 1 μg/mL pepS or pepN peptide pools. Cells stimulated with 4 μg/mL PHA served as positive controls. After combined surface (CD45, CD4, CD8, CD45RO, CD69) and intracellular cytokine staining (IFN-γ, TNFα, IL17A, granzymeB) specific T cell responses were acquired on a CytoFLEX Flow Cytometer (Beckman Coulter, California, US) and analysed with Flowjo v10.6 (Becton Dickinson, New Jersey, US). Antibodies and the respective suppliers are depicted in Table 2.

Fluorochrome labelled antibodies and suppliers.

(Biolegend, California, US; Tonbo Biosience, California, US).