Pe conjugated streptavidin antibody

Cell-surface expression of CD4 (BD Biosciences), CD8 (BioLegend), and CD171 (cat#130-100-691, Miltenyi Biotec) was detected by fluorophore-conjugated monoclonal antibodies. EGFRt expression was detected using biotinylated cetuximab (Bristol-Myers Squibb) and a phycoerythrin (PE)-conjugated streptavidin antibody (BioLegend). T cell activation and exhaustion were assessed by fluorophore-conjugated monoclonal antibodies detecting CD137 (BioLegend), CD25 (BioLegend), PD-1 (also known as PDCD1 or CD279, BioLegend), TIM3 (Biolegend), and LAG3 (BD Biosciences). Flow cytometry was performed on a Fortessa X-20 (BD Biosciences) and data were processed using FlowJo software (Tree Star Inc.). Dead cells were excluded from analyses using LIVE/DEAD^TM Fixable Green Dead Cell Stain Kit (Life Technologies).

Cell surface expression of L1CAM (clone REA163, Miltenyi Biotec), GD2 (clone 14.G2a; BD), CD3 (clone Hit3a, BioLegend, San Diego, CA, USA) and CD8 (clone SK1; BioLegend) was detected by fluorophore-conjugated monoclonal antibodies on a Fortessa X-20 (BD Biosciences, Franklin Lakes, NJ, USA) 4-laser flow cytometer. EGFRt expression was detected using biotinylated cetuximab (Bristol-Myers Squibb, New York, NY, USA) and a phycoerythrin (PE)-conjugated streptavidin antibody (cat #12-4317-87, BioLegend). Activation was assessed by fluorophore-conjugated monoclonal antibodies detecting TNFRSF9 (formerly CD137, clone 4B4-1; BioLegend) and IL2RA (formerly CD25, clone BC96; BioLegend). The Annexin V/7-AAD detection kit (BioLegend) was used to assess apoptosis. Dead cells were excluded from analyses using the LIVE/DEAD™ Fixable Green Dead Cell Stain Kit (cat#L23101, Life Technologies). Precision count beads (BioLegend) were used to quantify T cell infiltration according to the manufacturer’s instructions. Flow cytometry data was processed using FlowJo_V10 Software (Tree Star Inc., Ashland, OR, USA).