Figure 1. Anti-VEGFR2 CAR expression and ex vivo functional integrity of retrovirally engineered Tg-Pmel T cells.

A, CD3⁺ T cells from splenocytes of Wt or transgenic Pmel mice were stimulated with ConA and IL-7 or 1 μM hgp100_25–33 peptide respectively for 2 days in T cell media containing 30 IU /mL rhIL-2 and transduced with retroviral vectors expressing an anti-VEGFR2 CAR (DC101 CAR) or an empty vector. Two days later T cells were analyzed for expression of the DC101 CAR and Pmel TCR (measured by Vβ13 staining) by FACS. Cells were also costained for CD3ε expression using allo phycocyanin (APC) conjugated rat anti-mouse CD3ε. CD3⁺ viable T cells were gated. Representative FACS data from 3 experiments showing the percentage of cells in each quadrant are shown. B, Two days after transduction, 10⁵ effector mouse T cells were cocultured with indicated target cells at 1:1 ratio for 18 hours. Where indicated 2 effector T cell types were mixed in equal numbers (each 5 × 10⁴) and cocultured with 10⁵ target cells. Target cells were pulsed with indicated concentrations of either hgp100_25-33 peptide or irrelevant control peptide prior to coculture. Culture supernatants were assayed for secreted IFN-γ by ELISA. The data shown are representative of two independent experiments.