Anti ifn γ pe 4 s b3

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.

Virus-specific CD8⁺ T-cell frequencies were measured by flow cytometric analysis of gamma interferon (IFN-γ) induction after specific stimulation as described previously [42 (link)]. PBMCs were prepared from whole blood by density gradient centrifugation using Ficoll-Paque PLUS (Cytiva). Lymph node-derived lymphocytes were prepared from minced lymph nodes by density gradient centrifugation using Ficoll-Paque PLUS. Cells were pulsed and cocultured with peptide pools (at a final concentration of more than 0.1 μM for each peptide) using panels of overlapping peptides spanning the SARS-CoV-2 S, N, M, and E amino acid sequences (PM-WCPV-S-1, PM-WCPV-NCAP-1, PM-WCPV-VME-1, and PM-WCPV-VEMP-1; JPT Peptide Technologies) in the presence of GolgiStop (monensin, BD), 1 μg/ml of anti-CD28 (CD28.2, BD) and 1 μg/ml anti-CD49d (9F10, BD) for 6 hours. Intracellular IFN-γ staining was performed with a CytofixCytoperm kit (BD) and anti-CD3 APC-Cy7, anti-CD4 FITC, anti-CD8 PerCP, and anti-IFN-γ PE (4S.B3; BioLegend). Stained cells were analyzed by BD FACS Lyric. A representative gating schema for flow cytometric analysis is shown in Fig 5A. Specific T-cell frequencies were calculated by subtracting nonspecific IFN-γ⁺ T-cell frequencies from those after peptide-specific stimulation. Specific T-cell frequencies less than 0.03% of CD8⁺ T cells were considered negative.