Figure 2.

In Vitro Analysis

(A) Schematic of γ-retroviral vectors. See Figure S1A for details. Costimulatory domains were derived either from CD28 (CAR28) or 4-1BB (CAR41). (B) Cytolytic activity. CAR T cells were co-cultured at the indicated E:T ratios with PSMA⁺ C4-2 tumor cells. Cytotoxicity was determined using a cell viability assay (n = 3). (C) PSMA-mediated activation of CAR T cells. CAR T cells were co-cultured with PSMA⁺ (C4-2) or PSMA⁻ (Du145) tumor cells. T cell activation was assessed by evaluating expression of CD25. Shown is mean fluorescent intensity (MFI, n = 6). (D) Cytokine release. CAR T cells were co-cultured with PSMA⁺ (C4-2) or PSMA⁻ (Du145) cells and IFN-γ in supernatant was measured (n = 3). (E) CAR T cell phenotype. CAR T cells were co-cultured with PSMA⁺ tumor cells before the phenotype was assessed based on CD62L and CD45RA expression. Shown are the average percentages of the different T cell subsets (n = 3 or 4). (F) Exhaustion. CAR T cells were co-cultured with PSMA⁺ tumor cells and the extent of T cell exhaustion was assessed by measuring expression of CD223 (LAG-3). Shown are the average percentages of LAG-3⁺ cells (n = 3 or 4). ∗p < 0.05, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001. UT, untransduced T cells; Tn/Tscm, T cell naive or T stem cell memory; Tcm, T cell central memory; Tem, T cell effector memory; Teff, T cell effector; LAG-3, lymphocyte activation gene 3.