Apc conjugated anti cd8a antibody

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).

E0771 cells were stained with PE-conjugated anti-mouse CD284 (Biolegend, 117605) and Alexa Fluor 488-conjugated anti-mouse CD282 (Biolegend, 121807). To analyze CD8+ T cell infiltration in tumor tissues, E0771 tumors from WT and Bgn KO mice were dissected and digested with collagenase II and DNase I. Single-cell suspensions were stained with FITC-conjugated anti-CD45 antibody (Biolegend, 103115) and APC-conjugated anti-CD8a antibody (Biolegend, 100711). CD45 + CD8+ T cells were analyzed using a FACS Aria II. Data was analyzed using FlowJo V10 software (Tree Star Inc.).