Disruption of the TCR αβ-CD3 complex in primary T cells. (A) Schematic presentation of ZFN transfer. A pair of ZFN-encoding mRNA was electrotransferred 6 days after stimulation of CAR^neg T cells. T cells were then cultured with 50 IU/mL of IL-2 and incubated at 30°C or 37°C 5%CO₂, as indicated. CD3 expression was analyzed by flow cytometry on day 7 to 9 after electroporation. (B) Down-regulation of CD3 after electrotransfer of mRNA encoding the TCR αβ targeted ZFNs. Day 9 after electrotransfer of the indicated doses of mRNA coding for TRAC or TRBC targeted ZFN pairs, TCR αβ-CD3 expression was analyzed by costaining for CD4, CD8, and CD3ϵ. Representative flow data at day 9 after ZFN electrotransfer is shown. Flow cytometry data are gated on cells excluding PI. Numbers in the lower right quadrant represent the percentage of CD3ϵ negative cells in T-cell populations. Top panels show CD3ϵ expression in T cells cultured at 37°C after ZFN transfer and bottom panels show CD3ϵ expression in T cells transiently cultured at 30°C from day 2 to 3 after ZFN transfer. (C) Surveyor nuclease assay to detect ZFN-mediated modification of TCR target sites in T cells. 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 TRBC1 locus, respectively. Lane headings indicate both the mRNA dose, specific ZFN pair delivered via electrotransfer, and temperature of incubation for the different samples. Numbers beneath each lane indicate the percentage of modified alleles in each sample.