CAR expression and signaling in T cells with CD3/TCRαβ expression blockade. (A) Flow cytometric dot-plots illustrate CD3 downregulation and anti-CD19-41BB-CD3ζ CAR expression. Cells were transduced with the CAR construct followed by anti-CD3ε PEBL, or with GFP only followed by mCherry only (“Control”). (B) Percentage of T lymphocytes transduced with PEBL or GFP alone (“Control”) expressing anti-CD19-41BB-CD3ζ CAR 24 hours after CAR mRNA electroporation (n = 5), or 5 to 6 days after CAR viral transduction (n = 4); P = .207. (C) IFNγ production by PEBL or control T cells electroporated with CAR mRNA or no mRNA and cultured with CD19⁺ RS4;11 for 8 hours at E:T 1:2. Bars represent mean (±SD) of 9 measurements with cells from 3 donors; ****P < .0001. (D) T lymphocytes were first transduced with CAR and then transduced with either mCherry alone or anti-CD3 PEBL. Cells were then cultured with irradiated CD19⁺ OP-1 for 3 weeks. Results were compared with cells transduced with GFP only and then with mCherry only (“Control”). Symbols indicate mean (± SD) percentage cell recovery relative to number of input cells in triplicate cultures.