The functional response to target cell lines is significantly increased in CD8⁺ T cells cotransduced with TCR and CRISPR/Cas9 specific for endogenous TCR-β. (A) Polyfunctionality plots representing the response of transduced and untransduced T cells in comparison with the parental T-cell clone. Top row, The response to a B-LCL line preincubated with zoledronate by cells transduced with the γδ20 TCR. Bottom row, Responses to an HLA-A2⁺ melanoma cell line by cells transduced with the Mel13 αβ TCR. Only viable CD3⁺ cells were included in the analysis whereas the gates for cells positive for a given function were set based on appropriate fluorescence minus 1 and biological controls. Representative data from 2 independent experiments and 3 donors are shown. (B) The response of transduced T cells to target cell lines, in terms of CD107a, IFNγ, and TNFα expression (mean and standard deviation from 3 donors are shown). The percentage of cells that were positive for a given function in absence of cognate stimulus (ie, T cells plus B-LCL for γδ20 TCR, and T cells alone for Mel13 TCR) was subtracted from the percentage of cells positive in the presence of cognate stimulus (ie, T cells plus B-LCL preincubated with zoledronate or T cells plus HLA-A2⁺ melanoma cell line, respectively). The statistical significance of difference between the response of cells transduced only with TCR or with TCR plus CRISPR was measured by the paired Student t test. ***P = .0001; **P = .002.