Fitc conjugated cd4 antibody

Tumor tissues were digested with Liberase TM and Dnase I (Roche, Basel, Switzerland) and filtered through 40 μm cell strainers. Cells were initially incubated with CD16/32 antibody to block Fcγ receptors, then stained with 1) the combination of APC-Cy7-conjugated CD45 antibody (BD Biosciences, Franklin Lakes, NJ USA), FITC-conjugated CD4 antibody and APC-conjugated CD8a antibody (BioLegend, San Diego, CA USA), or 2) the combination of FITC-conjugated CD44 antibody, PE-Cy7-conjugated CD62L antibody and APC-conjugated CD4 or CD8a antibody (BioLegend). For Treg discrimination, cells were further fixed and permeabilized (eBioscience, San Diego, CA USA) according to manufacturer’s instructions, stained with PE-conjugated anti-FOXP3 antibody (eBiosciences), and analyzed with FACSAria cytometer (BD Biosciences).

The expression of EPHB4 was detected via phycoerythrin (PE)‐conjugated EPHB4 antibody (R&D Systems, Inc., Minneapolis, MN, USA) staining. For the detection of EPHB4‐CAR expression, transduced T cells were stained using goat anti‐Ephrin B2 antibody (R&D Systems) and then stained using PE‐conjugated anti‐goat IgG antibody (R&D Systems). Allophycocyanin (APC)‐conjugated anti‐CD3 antibody, APC‐conjugated anti‐CD8a antibody, fluorescein isothiocyanate (FITC)‐conjugated CD4 antibody, FITC‐conjugated anti‐CD45RA antibody, and APC‐conjugated anti‐CCR7 antibody (all from BioLegend) were used for the characterisation of the CAR‐T cell phenotype. To determine the binding capacity of human Ephrin B2 to cynomolgus EPHB4, 293‐human EPHB4‐GFP and 293‐cynoEPHB4‐GFP cells were incubated with the recombinant human Ephrin B2‐Fc Chimera Protein (R&D Systems) for 20 min on ice and then stained using APC‐conjugated anti‐human IgG‐Fc antibody (BioLegend). Detailed antibody information is presented in Supplementary table 2. All flow cytometry data were acquired using BD FACS Accuri C6 Plus (BD Biosciences) and analysed using the FlowJo Software (Tree Star, Inc., Ashland, OR, USA).