To allow for Spike binding on the cell surface, CD8+ T cells (0.5×106) were incubated for 30 min at 20°C in 50 μl RPMI-HEPES in the absence of BCS with 2.5 μg Spike Wuhan/BA.1/BA.2 or PBS vehicle control (concentration of Spike treatment chosen based on a dose-response analysis; see Figure S2B ). Alternatively, CTLs were pre-treated under the same conditions with 1.2×109/ml MiniVs or control liposomes. Spike-W and the Omicron BA.1 variant were also incubated in combination with the respective neutralizing mAbs (J08 and 02M04, respectively) at a 1:3 protein:antibody ratio. Alternatively, CD8+ T cells (0.5×106) were incubated for 30 min at 20°C in 50 μl RPMI-HEPES in the absence of BCS with 2 μg/ml anti-ACE2 antibody (R&D Systems, #AF933) (Hoffmann et al., 2020 (link)), or the ACE2 substrate/ligand Angiotensin II (Merck, #A9525; 1 μM), or the ACE2 product Ang (1-7) (Sigma, #A9202; 1 μM).
For cytotoxicity assays, cells were pre-treated in the same conditions in serum-free AIM V medium (Gibco, #12055-091) (calcein release assays) or RPMI 1640 (flow cytometry-based assays). None of the treatments affected cell viability at the concentrations and times chosen for the analyses, as assessed by Trypan blue (Sigma-Aldrich, T8154) exclusion (see legend tofigures 3 and 5 ).
For cytotoxicity assays, cells were pre-treated in the same conditions in serum-free AIM V medium (Gibco, #12055-091) (calcein release assays) or RPMI 1640 (flow cytometry-based assays). None of the treatments affected cell viability at the concentrations and times chosen for the analyses, as assessed by Trypan blue (Sigma-Aldrich, T8154) exclusion (see legend to
