Anti ifnγ mab

Freshly thawed (ex vivo) PBMCs from healthy donors were analyzed by enzyme-linked immunospot (ELISPOT) assay in duplicates. Interferon γ (IFNγ) ELISPOT assays in our study were performed as described previously^{49 (link)}. In brief, 96-well nitrocellulose plates (Millipore) were coated with 1 mg/mL anti-IFNγ mAb (Mabtech) and incubated overnight at 4 °C. In a next step, plates were blocked with human serum (10%) for 2 hours at 37 °C. PBMCs (2.5 × 10⁵ cells per well) were pulsed with an EBV/CMV epitope mix containing the frequently recognized peptides BRLF109-117 YVLDHLIVV (A*02) peptide and CMV pp65 (A*02) peptide NLVPMVATV and incubated with or without platelets (ratio 1:50) for 24 h. Phytohemagglutinin was used as positive control. HLA-A*02 (KLFEKVKEV)- and B*07 (KPSEKIQVL)-restricted control peptides derived from benign tissues (HV-exclusive HLA ligands) served as negative control. Prior co-incubation with T cells PD-L1 positive platelets from NSCLC patients were pre-treated with the anti-PD-L1 mAB Atezolizumab for 30 min and washed twice with PBS containing 1% FCS. Readout was performed according to the manufacturer’s instructions. Spots were counted using an ImmunoSpot S5 analyzer (CTL).