Evaluation of EGFRvIII/CAR expression on CD3+T cells. (A) Schematic representation of EGFRvIII/CAR. It consists of EGFRvIII scFv, the hinge and transmenbrane (TM) region of human CD8α, ICOS signaling domain, and human CD3ζ chain. IgG κ chain was used as signal peptide (SP). (B) Surface expression of EGFRvIII/CAR on CD3+ T cells. Left, isotype control; Middle, anti-human CD3-FITC and anti-human CD8-PE staining (BD Biosciences); Right, anti-mouse F(ab)2-FITC (eBiocience) and anti-human CD8-PE staining. (C) Immunoblot analysis of EGFRvIII/CAR expression. Lysates of untransduced T cells (lanes 1) and EGFRvIII/CAR transduced T cells (lane 2) were separated by SDS-PAGE under reducing condition. Goat anti-human CD3ζ antibody was used to detect the endogenous and chimeric CD3ζ expression.