Jes6 1a12

IL-2 and IFN-γ production by T cells were detected using BD Biosciences recommended ELISA protocols. IL-2 and IFN-γ from cell supernatants were captured by rat anti-mouse IL-2 (JES6-1A12, BD Biosciences) and rat anti-mouse IFN-γ (R4-6A2, BD Biosciences), respectively. Detection of the captured cytokines was accomplished through the use of biotinylated rat anti-mouse IL-2 (JES6-5H4, BD Biosciences) and biotinylated rat anti-mouse IFN-γ (XMG1.2, BD Biosciences) antibodies. Graded amounts of recombinant murine IL-2 and IFN-γ were included for generation of standard curves from which concentrations were extrapolated using the linear portion of each curve. Reported IL-2 and IFN-γ values were acquired through the use of a BioTek Eon microplate spectrophotometer (BioTek Instruments Inc., Winooski, VT, USA) and analyzed using Gen 5 software version 2.01.14.

Activation of human FcγRIIIA (CD16) was assessed using a surrogate activation assay^{37 (link)}. Briefly, IgG-dependent activation of BW:FcγRIIIA-ζ transfectants, i.e. BW5147 thymoma cells (TIB-47TM ATCC; Virginia, USA), expressing the extracellular portion of human FcγRIIIA (higher affinity variant with valine in position 158) fused to the mouse CD3 ζ–chain, was measured. SARS-CoV-2 S1 and N proteins were coated on plates using coating buffer (0.1 mol/L Na₂HPO₄, pH 9.0). After blocking with 5% fetal calf serum (FCS), 20 mg/dL of either anti-S1, anti-Mix, or convalescent human plasma in DMEM 10% (v/v) FCS was added for 30 min at 37 °C in an atmosphere of 5% CO₂. To remove non-immune IgG, plates were washed three times with DMEM containing 10% (v/v) FCS. Then, 100 000 BW:FcγRIIIA-ζ reporter cells per well in RPMI 10% (v/v) FCS medium were added. After co-cultivation for 16 h at 37 °C in a 5% CO₂ atmosphere, supernatants were diluted 1:2 in ELISA sample buffer (PBS with 10% [v/v] FCS and 0.1% [v/v] Tween-20) and mIL-2 was measured by ELISA using the capture Ab JES6-1A12 and the biotinylated detection Ab JES6-5H4 at 450 nm (BD Pharmingen™; San Diego, USA). Experiments were performed in triplicate.

The BW5147:FcγR-ζ reporter assays were performed as a surrogate test for FcγR activation^{36 (link),37 (link)}. Briefly, the assessment of IgG-dependent activation of the BW5147:FcγR-ζ cells was performed by incubating MCMV-infected cells with serial dilutions of serum in DMEM containing 10% (v/v) FCS for 30 min at 37 °C in an atmosphere of 5% CO₂. To remove non-immune IgG, cells were washed three times with DMEM containing 10% (v/v) FCS before co-cultivation with BW5147:FcγR-ζ reporter cells was performed in RPMI containing 10% (v/v) FCS. The ratio between BW5147:FcγR-ζ and infected cells was 20:1. After 16 h of co-cultivation, supernatants were diluted 1:2 in ELISA sample buffer (PBS with 10% [v/v] FCS and 0.1% [v/v] Tween-20) and mIL-2 was measured by ELISA using the capture antibody JES6-1A12 and the biotinylated detection antibody JES6-5H4 (BD Pharmingen, dilution 1:500).

FACS-purified subsets of CD8⁺ T cells from mice indicated were labelled with CFSE (2 μM) as described previously12 (link) and cultured for 2–7 days either with high doses of cytokines, IL-2 (0.1–1 μg ml⁻¹) and IL-7 (1 μg ml⁻¹), or with Cx-αCD3 mAb (0.1–10 μg ml⁻¹); in some experiments with the latter TCR stimulation, a mixture of mAbs to IL-2 (JES6-1A12) and CD122 (TM-β1; 10 μg ml⁻¹; all from BD Biosciences) were added to block IL-2 signalling. Cells were collected and CFSE dilutions were analysed by flow cytometry. Alternatively, proliferative responses of the indicated TCR-stimulated CD8⁺ T cells were analysed by adding [³H]thymidine (1 μCi per well) and after a 6–12 h pulse, cells were collected and measured by a β-counter (TopCount Microplate Scintillation Counter; PerkinElmer).