Elimination of TCR αβ-CD3 complex from CD19-specific CAR+ T cells. (A) Schematic of electrotransfer of mRNA coding for ZFN pairs in CAR+ T cells. mRNA species encoding the indicated ZFN pairs were electrotransferred into CAR+ T cells 2 days after stimulation with CD19+ aAPC. After electroporation, cells were maintained with 50 IU/mL of IL-2 and incubated for 2 days at 30°C 5%CO2. CD3ϵ expression was analyzed 9 days after electroporation by flow cytometry. (B) Disruption of TCR αβ-CD3 complex expression after electrotransfer of mRNA encoding the TCR-specific ZFNs. Flow cytometry analysis of CD3ϵ expression in T cells 9 days after electrotransfer of mRNA species encoding the indicated ZFN pairs, gated on the PI-negative population. (C) Surveyor nuclease assay. Arrows indicate the fragments produced by a surveyor nuclease digest of amplicons bearing a mismatch at the intended site of ZFN cleavage in the TRAC or TRBC loci, respectively. Samples were analyzed 9 days after electroporation. The numbers at the bottom represent percentages of modified alleles in each sample.
