L1cam clone rea163

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).

Cell surface expression of L1CAM (clone REA163, Miltenyi Biotec, Bergisch Gladbach, Germany), GD2 (clone 14.G2a; BD), HER2 (clone 24D2, Biolegend, San Diego, CA, USA) and CD8 (clone SK1, BioLegend) was detected by fluorophore-conjugated monoclonal antibodies. EGFRt expression was detected using biotinylated cetuximab (Bristol-Myers Squibb) and a phycoerythrin (PE)-conjugated streptavidin antibody (cat #12-4317-87, BioLegend). The APC Annexin V Apoptosis detection kit (cat #422201, BioLegend) was used to assess apoptosis after 24, 48 and 72 h of stimulation with L1CAM⁺ tumor cells (E:T ratio, 1:5). Dead cells were excluded from analyses using LIVE/DEAD^TM Fixable Green Dead Cell Stain Kit (cat #L23101, Life Technologies). Cell surface expression from murine T cells was identified using Cd8 (clone 53-6.7, BD Bioscience, San Jose, CA, USA), Pd1 (clone J44, BD Bioscience) and Tim3 (clone RMT3-23, BD Bioscience). Flow cytometry was performed on Fortessa X-20 (BD Biosciences) and FACSCanto II (BD Biosciences) instruments. Data was processed using FlowJo_V10 Software (Tree Star Inc., Ashland, OR, USA).