Live dead zombie aqua fixable viability kit

Cells were washed in FACS buffer (PBS + 2% FBS + 2 nM EDTA) and then stained for 30 min on ice with a cocktail containing fluorophore-conjugated antibodies diluted in FACS buffer. The antibody cocktail consisted of: APC/Cy7-CD3 (SK7, Biolegend, San Diego, CA, USA), PE/Cy7-CD4 (A161A1, Biolegend), APC-CD8 (SK1, Biolegend), FITC-CD24 (HSA, M1/69, BD Biosciences), and the LIVE/DEAD Zombie Aqua Fixable Viability Kit (Biolegend). Stained cells were washed twice with FACS buffer and then fixed with 2% paraformaldehyde for 20 min. UltraComp eBeads Compensation Beads (ThermoFisher) were used for each antibody individually for use as compensation controls, per the manufacturer’s protocol. Flow cytometric samples were run on a LSRFortessa (BD Biosciences, San Jose, CA, USA) and infection rates in CD4 + T cells were quantified using FlowJo software (FlowJo LLC, BD, Ashland, OR, USA). Productively-infected CD4+ T cells were identified as live, CD3+CD8- cells expressing the reporter gene HSA and that have downregulated cell-surface CD4.

Cryopreserved PBMCs from 21 CHIRP participants were thawed and cultured overnight to enable antigen recovery, and then screened by FACS for specific binding to APC-labeled MHC class I tetramers (Table S2) obtained from the NIH Tetramer Core Facility. These tetramers harbor predicted MHC class I epitopes from SARS-CoV-2. For tetramer staining, 1 million cells were transferred into 96-well V-bottom polystyrene plates and washed once with FACS buffer (PBS supplemented with 2% FBS and 2 mM EDTA), stained with 15 μg/ml the APC-labeled MHC class I tetramer for 1h at room temperature. Cells were then washed twice with FACS buffer, and stained for 30 min on ice with a cocktail of antibodies diluted in a 1:1 mixture of FACS buffer and the Brilliant Stain Buffer (BD Biosciences). The antibody cocktail consisted of APC/Cy7-CD3 (SK7, Biolegend), BD Horizon^™ BV650-CD8 (RPA-T8, BD Biosciences), BD Horizon^™ BUV737-CD4 (SK3, BD Biosciences), and the LIVE/DEAD Zombie Aqua^™ Fixable Viability Kit (Biolegend). After 3 additional washes with FACS buffer, cells were fixed and analyzed on an LSRFortessa^™ (BD Biosciences).