Figure 4.
Production of B7-H3-Redirected CAR-T Cells
(A) Schematic representation of the B7-H3-targeted CAR construct containing the following fragments: J42-scFv; hinge; CD8 transmembrane domain; intracellular signaling domains of CD28, 4-1BB, and CD3-ζ; P2A; and mCherry. (B) B7-H3 CAR expression in human T cells was analyzed by tracking mCherry expression with FACS. (C and D) 10 days after lentivirus CAR transduction, the subsets and phenotype of NT and CAR-T cells were analyzed by FACS, including the expression of (C) CD4, CD8, CD45RO, CD62L and (D) PD1, TIM3, CD25, CD69. (E) Immunofluorescence staining for the expression of B7-H3 (red or green) in A375, MV4-11, and Raji cells. Scale bars, 10 μm. (F) 51Cr-release assay to measure the cytotoxicity of CAR-T cells against A375, MV4-11, and Raji cells at different E:T ratios. All error bars represent SD. ∗p < 0.05.