Cd4 a700

PBMCs were harvested after 24 h co-incubation with ONCOS-204 infected A375 cancer cells and 0.05 ng/ml EGFRxCD3 BsAb. After washing, and staining with viability dye, PBMCs were stained with EGFR-PE (BD), CD3-PerCP (Biolegend), ICOSL-A647 (BD), CD4-A700 (BD), CD8-APC/Cy7 (BD), ICOS-BV421 (BD) and CD69-BC785 (Biolegend).

The following monoclonal antibodies were purchased from eBioscience: CD278 (ICOS)-biotin, CD27-PeCy7, Foxp3-FITC, RORγt-PE, T-bet-PE; or from BD Biosciences: PD1-PECF594, CXCR3-APC, CD24-PECF594, CD25-BB515, CD44-PECy7, CD4-A700, CD8-A700, CD4-PB, CD62L-A700, GATA3-PE, RORγt-PECF594, STAT1 (pY701)-A488, IFNγ-PE, IL-10-APC, IL-17-PerCP-Cy5.5, streptavidin-PECy7. Live/dead fixable near-IR stain (Thermo Fisher) was used to exclude dead cells. For transcription factor staining, cells were stained for surface markers, followed by fixation and permeabilization before nuclear factor staining according to the manufacturer’s protocol (Foxp3 staining buffer set from eBioscience). For cytokine staining, cells were stimulated in media containing phorbol 12-myristate 13-acetate (50 ng/ml, Sigma-Aldrich), ionomycin (250 ng/ml, Sigma-Aldrich), and brefeldin-A (1/100, eBioscience) for 3 hr. After stimulation, cells were stained for surface markers, followed by fixation and permeabilization before intracellular staining according to the manufacturer’s protocol (cytokine staining buffer set from BD Biosciences). For phosphorylation staining, cells were stimulated with IFN-γ (50 ng/ml, PeproTech) for 30 min, fixed with formaldehyde, and permeabilized with methanol before staining. Flow cytometric analysis was performed on a Canto II (BD Biosciences) and analyzed using FlowJo software (Tree Star).