CAR constructs and expression of CD27 containing FR CAR or CD19 CAR on primary human CD4 and CD8 T cells. (A) Schematic representation of anti-FR MOv19 scFv-based CAR constructs containing the CD3ζ cytosolic domain alone (FR-z CAR) or in combination with CD27 intracellular domain (FR-27z CAR). CD19-27z CAR was also constructed and used as an antigen specific control. VL indicates variable light chain; L, linker; VH, variable heavy chain; and TM, transmembrane region. (B) Both primary human CD4 and CD8 T cells can efficiently express either FR-specific CARs or CD19-specific CAR as measured by flow cytometry. A viable CD3⁺ lymphocyte gating strategy was used. UNT indicates untransduced cells.