Permeabilizing buffer

Approximately 1.5 × 10⁶ cells were stained with antibodies and antibody application was followed by the recommendation. Mouse lymphocytes were stimulated with the peptide pool of SARS-CoV-2 RBD and incubated with monensin [BioLegend (Cat. No. 420701)] for 9 hours. Then, the cells were harvested. For surface staining, cells were stained with fluorescence-labeled mAbs of CD3-FITC (BioLegend, USA), CD4-APC-Cy7 (BioLegend, USA) and CD8-AF700 (BioLegend, USA). The cells were subsequently fixed and permeabilized in permeabilizing buffer (BD Biosciences, USA) and intracellularly stained with fluorescence-labeled mAbs of IFN-γ-BV605 (BioLegend, USA), IL-2-BV421 (BioLegend, USA) and IL-4-PE (BioLegend, USA). All stained cells were detected on BD LSRFortessa™ X-20.

The ICS assay was carried out exactly as previously described^{30 (link),45 (link),46 (link)}. Briefly, TCR-transduced T cells were co-cultured in 96-well plates with target cells for 2 h. As negative and positive controls, cells grown with medium alone or PMA/ION (Dakewe Biotech Co., cat. 2030421) were utilized. For a further 6 h at 37 °C, the cells were treated with GolgiStop (BD Biosciences, cat. 555029). Anti-CD3 and anti-CD4/or anti-CD8 surface markers were used to label cells before they were fixed and permeabilized in permeabilizing buffer (BD Biosciences, cat. 51-2090KZ) and stained with anti-IFN-γ-PE (4 S.B3, Biolegend, cat. 502509). Following washes and re-suspension, samples were examined on a FACS Aria II.

For ICS assay, fresh mouse splenocytes were added into 96-well plate (1 × 10⁶ cells/well). Cells were stimulated with each peptide pool (2 μg/ml for each peptide) for 3 h, and incubated with Golgiplug (BD Biosciences, USA) for 6 h at 37°C. Then, cells were harvested, blocked with recombinant CD16/32 protein, and stained with PE/Cy7-conjugagted anti-CD3, APC/Cy7 conjugated anti-CD4 and PerCP/Cy5.5 conjugated anti-CD8α antibodies (Biolegend, USA). After fixation and permeabilization by permeabilizing buffer (BD Biosciences, USA), cells were further stained with PE conjugated anti-IFN-γ antibody (Biolegend, USA). Cells were analyzed by flow cytometric analysis through a BD LSRFortessa flow cytometer (BD Biosciences, USA) with high-throughput system. Data were analyzed using FlowJo 10.0 (BD Biosciences, USA).

CD4⁺ and CD8⁺ T‐cell responses were evaluated using ICS. Briefly, mouse splenocytes were added to a cell plate (1 × 10⁶ cells/well) and then stimulated with the peptide pool (2 μg/ml of individual peptides) for 4 h. Dimethyl sulfoxide (40%) and phorbol 12‐myristate 13‐acetate/ionomycin were used as negative and positive controls, respectively. The cells were incubated with GolgiPlug (BD Biosciences) for an additional 12 h at 37°C. The cells were washed with phosphate‐buffered saline (PBS) and surface‐stained with anti‐mouse CD3/CD4/CD8 antibodies (BioLegend) for 30 min at room temperature. The cells were subsequently fixed, permeabilized in permeabilizing buffer (BD Biosciences), and stained with fluorochrome‐labeled antimouse IFN‐γ/TNF‐α/IL‐4antibodies (BioLegend). An LSRFortessa flow cytometer (BD Biosciences) was used to acquire the flow cytometry data, which were then analyzed using FlowJo software (Becton Dickinson).

Intracellular cytokine staining assay was performed as previously described (53 (link)). Briefly, TCR-transduced T cells were co-cultured with SCT-K562 cells at a ratio of 1:1 in 96-well-plates for 4 h. Cells cultured with medium alone or PMA/ION were used as negative or positive controls, respectively. The cells were incubated with Golgistop (BD Biosciences, San Jose, California) for an additional 10 h at 37°C. Cells were stained with anti-CD3 and anti-CD4/or anti-CD8 surface markers, fixed, and permeabilized in permeabilizing buffer (BD Biosciences, San Jose, California), and subsequently stained with anti-IFN-γ-PE/Cy7. Samples were then washed, re-suspended, and then analyzed on a FACSAria III.