Fig. 4. FOXP3 editing of T_eff cells preserves physiological regulation of FOXP3 expression and in vitro function.

(A) Flow cytometry time course showing kinetics of FOXP3 expression in nonactivated T_eff cells and activated T_eff cells on subsequent days after activation [day 3 (d3), d6, and d14], comparing WT unmodified, WT mock-treated, and FOXP3^FLcoW edited T_eff cells. FOXP3 expression quantified in T_eff cells over time course of activation, showing average MFI (mean ± SD, n = 3). (B) Cytokine production in WT and FOXP3 gene edited T_eff cells determined by enzyme-linked immunosorbent assay (ELISA). Supernatants collected at 24 hours (IL-2) and 48 hours (IFN-γ and IL-17) after activation with anti-CD3/28 (mean ± SD, n = 3). (C) T_eff cell proliferation in response to activation measured by the proliferation assay. Flow cytometry plots of carboxyfluorescein diacetate succinimidyl ester (CFSE) dye–stained T_eff cells with progressive dilution of dye as proliferation progresses from nonactivated to day 2 and day 3 after activation with anti-CD3/28 Dynabeads are shown. Comparison of proliferation rates in response to activation with a bead:cell ratio of 1:100 and 1:25. Quantification of average proliferative response of T_eff cells from proliferation assay at day 3 is shown to the right, comparing different doses of activation beads (mean ± SD, n = 3).