Icos bv421

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).

To determine the NK cells and lymphocyte subsets in patients, heparin‐anticoagulated whole blood samples were collected and stained with (1) CD3‐PerCP (BD Biosciences, San Jose, CA,), CD4‐BV510 (BD Biosciences, San Jose, CA), CD8‐APC (BD Biosciences, San Jose, CA), CD45RO‐BV421 (BioLegend, San Diego, CA), and CCR7‐PE (BD Biosciences, San Jose, CA); (2) CD4‐APC‐Cy7 (eBioscience, San Diego, CA), CXCR5‐APC (eBioscience, San Diego, CA), TIM‐3‐PerCP (eBioscience, San Diego, CA), TIGIT‐FITC (eBioscience, San Diego, CA), CD226‐PE‐Cy7 (eBioscience, San Diego, CA), PD‐1‐BV510 (BD Biosciences, San Jose, CA), and ICOS‐BV421 (BD Biosciences, San Jose, CA); (3) CD19‐PE (eBioscience, San Diego, CA), CD27‐PE‐Cy7 (eBioscience, San Diego, CA), CD38‐APC (eBioscience, San Diego, CA), CD24‐APC‐Cy7 (eBioscience, San Diego, CA), IgM‐BV421 (eBioscience, San Diego, CA,), and IgD‐FITC (eBioscience, San Diego, CA); and (4) CD3‐FITC (BD Biosciences, San Jose, CA) and CD16 + CD56‐PE (BD Biosciences, San Jose, CA). Cell counting was performed with absolute counting tubes with a certain number of beads (BD Biosciences, San Jose, CA). The samples were measured using FACS Canto II (BD Biosciences, San Jose, CA). Gating analysis was performed with FlowJo V10 (BD Biosciences, San Jose, CA).