Apc anti human cd3

All flow cytometry measurements were carried out using a BD Accuri C6 device. Cells were stained for 45–60 min at 4°C–8°C. T cell activation was assessed by staining with anti-human CD3-APC (Miltenyi Biotec, clone BW264/56) and anti-human CD25-PE (Miltenyi Biotec, clone 4E3). TRAC knock-out was assessed 6–7 days after gene editing by staining with anti-human TCRα/β-PE (Miltenyi Biotec, clone BW242/412) and anti-human CD3-APC (Miltenyi Biotec, clone BW264/56).

96 h after Jurkat cell transduction with LeGO-CCR5-iB2-Puro+ or LeGO-(CCR5^Δ55-60)-iB2-Puro+, cells were harvested, washed with PBS, and resuspended in 250 µl PBS. Cells were fixed and permeabilised with the Inside Stain Kit (Miltenyi Biotec). Staining of cells was performed using 5 µl of the PerCP/Cy5.5 anti-human CD195 (BioLegend, San Diego, CA) and 5 µl of APC anti-human CD3 (Miltenyi) antibodies. After 15 min incubation in the dark at room temperature, cells were washed with PBS and resuspended in 100 µl fresh PBS. Cell images were obtained using the ImageStreamX Mk II System (Amnis/Luminex, Austin, TX); data were acquired and analysed with IDEAS Software package (Amnis/Luminex) using channels 5, 7, 11 and brightfield. Compensation was performed according to the software introduction using single-stained cells. BFP- (CCR5, or CCR5^Δ55-60) positive cells were gated and their images investigated. Normal erode masque was applied to all images, and the internalization wizard was used to check the relative BFP to Cy5.5 signal localisation (Internalisation of BFP signal by Cy5.5 signal marking CCR5). Cells with internalised BFP signals were selected by choosing the cell population with an internalisation score ≥1.

The expression of human hGMR‐CAR T cells on the cell surface was determined using APC anti‐human CD3 (Miltenyi Biotec), APCcy7 anti‐human CCR7 and APCcy7 anti‐human CD8 (BioLegend). All other reagents were the same as those used in the cynomolgus phenotypic analysis.