T-cell ELISpot assay

ELISpot plates (MilliporeSigma) were coated with either IFNγ (BD Biosciences, #51–2525KZ), IL-17A (Invitrogen, #88–7371–88), or IL-5 (BD Biosciences, #51–1805KZ) capture antibodies at a 1:200 dilution in Dulbecco’s PBS (DPBS; Gibco). After overnight incubation at 4 ^oC, plates were washed and then blocked with complete RPMI (cRPMI) medium for at least 2 h. Splenocytes harvested as described above were plated at 2 × 10⁵ cells per well. A subset of each sample was stimulated with PepMix SARS-CoV-2 (JPT Peptide Technologies, #PM-WCPV-S-1) at a final concentration of 1 µg/mL. Plates were then incubated at 37 °C and 5% CO2 for 48 h. After a wash with PBS with 0.1% Tween 20, 100 µL of detection antibody (IFNγ, BD Biosciences, #51–1818KA; IL-17A, Invitrogen, #88–7371–88; and IL-5, BD Biosciences, #51–1806KZ) was added at a 1:250 dilution in ELISpot diluent (eBioscience) overnight at 4 °C. Plates were washed and developed with Vector NovaRED Substrate Peroxidase (Vector Laboratories, Burlingame, CA; #SK-4800) for 15 min. The reaction was stopped by washing the plates with deionized water, and plates were left to dry in the dark. Spots were counted and data were analyzed using ImmunoSpot 7 software (Cellular Technology Limited, Cleveland, OH).